Ultrasound Assessment of Aortic and Carotid Artery Intima-media Thickness in Children and Adolescents

Summary

The present article describes the methodological considerations for the noninvasive assessment of abdominal aortic and carotid intima-media thickness using B-mode ultrasonography. This technique is commonly used in the developmental origins of health and disease research as a surrogate for early arterial changes.

Abstract

Carotid intima-media thickness (IMT), measured using high-resolution B-mode ultrasonography, is a widely utilized surrogate marker of subclinical atherosclerosis, the pathophysiological process underlying most clinical cardiovascular disease events. Atherosclerosis is a gradual disease that originates early in life, thus, there has been increased interest in measuring carotid IMT in childhood and adolescence to assess structural change in the arterial vasculature in response to adverse exposures. However, the timing of atherosclerosis varies across the vascular tree. Primordial atherosclerotic lesions are present in the abdominal aorta as early as infancy, compared to mid-adolescence for the common carotid. Measurement of IMT at either site is susceptible to several technical challenges that need to be considered, especially in younger children. In this paper, we provide a detailed stepwise method for high-quality assessment of IMT of the abdominal aorta and common carotid artery in the young. We also provide insight into the appropriateness of either site when exploring the associations between early-life exposures and later-life cardiovascular disease.

Introduction

The Developmental Origins of Health and Disease (DoHAD) hypothesis proposes a link between environmental exposures during critical periods of development - from conception to 2 years of age - and later-life susceptibility to cardiometabolic diseases¹. Several observational studies have shown that exposures in the perinatal period, such as low birth weight and pre-term birth, are associated with longer-term cardiovascular disease (CVD) risk². Atherosclerosis, the gradual thickening of the two innermost layers of the arterial wall, is a precursor to most clinical CVD events³. This thickening can be measured non-invasively at the sub-clinical stage using high-resolution Brightness mode (B-mode) ultrasound, a technique referred to as intima-media thickness (IMT).

In the 1980s, carotid IMT measured ultrasonography was validated against direct histology and since then has become a hallmark non-invasive method to identify early arterial changes⁴. Assessment of carotid IMT is popular within DoHAD research as it allows us to explore the association between environmental exposures and adaptations in the vasculature early in life and the potential monitoring of these adaptations over time. Carotid IMT is increased in children with exposure to early-life risk factors such as fetal growth restriction⁵, and excessive weight gain in the first two years of life⁶, in addition to traditional CVD risk factors such as obesity⁷, smoking exposure, and dyslipidemia⁸. While IMT of the carotid bifurcation, internal, and common carotid have been studied with risk factors and are all predictive of later-life cardiovascular events^9,10, far-wall IMT of the common carotid (cIMT) artery is the only site to have been validated against direct histology³ and the focus of the present manuscript.

Importantly, studies exploring the natural progression of atherosclerosis indicate that the abdominal aorta is the first of the large elastic arteries to present with primordial atherosclerotic lesions known as fatty streaks, particularly the distal far wall of the vessel^11,12. Comparatively, the common carotid presents with fatty streaks in mid-adolescence. Thus, measurement of abdominal aortic IMT (aIMT) may facilitate earlier detection of changes in vascular structure. In the Muscatine Offspring Study of 635 people aged 11 - 34 in the United States, aIMT was found to have stronger associations with conventional CVD risk factors in adolescents (11-17 years), while cIMT had stronger associations in older subjects (18-34)¹³. In high-risk children compared to controls, IMTs of both vessels increased, but the effect was greater in the aorta compared to the carotid, accounting for luminal diameter¹⁴. These results and natural history studies collectively suggest prioritizing the measurement of aIMT in younger populations compared to cIMT. Although this is not without its limitations, the measurement of aIMT tends to be more variable¹⁵, the methodology until recently lacked standardization³, and there are concerns about its utility in individuals with greater central adiposity.

When focusing specifically on exposures in the first 1,000 days of life, two recent systematic reviews and meta-analyses provide meaningful insights into the sensitivity of each technique. In studies with apparently healthy subjects aged 0 to 18, Epure et al.⁹ assessed the associations between clinical conditions within the first 1,000 days of life and cIMT. They found being born small-for-gestational age (SGA), with or without fetal growth restriction, was significantly associated with increased cIMT in children and adolescents (16 studies, 2,570 participants, pooled standardized mean difference 0.40 [95% CI: 0.15-0.64], p= 0.001, I² = 83%) compared to those born appropriate for gestational age. In a near-identical meta-analysis with aIMT instead as the outcome measure, Varley et al.¹⁰ reported significantly increased aIMT for those born SGA compared to controls and the magnitude of the effect was greater than that for cIMT (14 studies, 592 participants, pooled standardized mean difference 1.52 [95% CI: 0.98-2.06], p < 0.001, I² = 97%). Moreover, they found associations with other risk factors that Epure et al.⁹ did not, such as exposure to pre-eclampsia and being born large-for-gestational age, perhaps owing to the greater sensitivity of aIMT than cIMT.

Importantly, both reviews identified a lack of standardization in methodology and an absence of tailored advice for measuring children and adolescents as a limitation for in-depth cross-study comparisons and inconclusive results for other exposures. Accordingly, the present manuscript aims to provide a detailed protocol for each measurement in the young. The rationale and justification for these protocols have been presented in greater detail previously³. We discuss common methodological challenges and provide practical recommendations to overcome them.

The below protocol assumes a basic understanding of an ultrasound machine and its components and the manipulations that can be performed with an ultrasound transducer^16,17,18. It is also strongly recommended that the examiner, participant, and machine are positioned appropriately to increase the efficiency of testing and minimize strain, consistent with modern best practices in sonography. Suggestions are provided below. All assessments should be performed in a quiet, temperature-controlled room with dimmed lighting for the comfort of the participant and the ascertainment of imaging. Ask participants to fast for at least eight hours before testing to reduce gas in the bowel¹⁵, clear fluids are allowed, although at younger ages this may not be possible. However, avoid assessing straight after a meal. Conducting measurements in the morning within the first two hours of awakening has been previously reported as the best timeframe for visualization of the abdominal aorta^3,15, this may also reduce any inconvenience associated with fasting. This protocol has been adapted from guidelines outlined by the American Society of Echocardiography Carotid Intima-Media Thickness Task Force¹⁹, the Mannheim Carotid Intima-Media Thickness and Plaque Consensus¹⁸, and the Association for European Paediatric Cardiology AECP²⁰ for the measurement of cIMT and recently published recommendations for the measurement of aIMT³. We strongly recommend also reviewing a recent point-of-care ultrasound protocol to assist with understanding the anatomy of the abdominal aorta and surrounding structures²¹.

Protocol

All research was performed in compliance with the Sydney Local Health District Human Research Ethics Committee (Protocol Nos. X16-0065 and X15-0041). All ultrasound images are free of identifying information. Images used to illustrate transducer placement were performed on individuals with their consent or with consent from their parent or guardian for those unable to provide consent.

1. Common carotid intima-media thickness

1. Ask the participant to lie down on the bed without a pillow. This will help keep the neck as straight as possible.
2. Ask the examiner to be positioned at the head of the participant and the bed elevated to allow the examiner's elbows to rest on the bed to stabilize the scanning arm.
3. Place the machine in front of the examiner, opposite the scanning side, to manipulate the machine with the examiner's non-scanning hand without overextension. Allow sufficient space so that the ultrasound can be moved to the opposite side when examining the other side of the neck.
4. Acquire an electrocardiogram (ECG) simultaneously. A 3-lead ECG is sufficient; apply the appropriate leads as per manufacturer instructions and the age of the participant. Place gel on the transducer. In neonates and infants, the use of single-use sterile gel packets is recommended for infection control purposes. Warming the gel beforehand may also help prevent discomfort.
5. Using a linear transducer with a minimum frequency of 7 MHz, set the device to a depth of 3-4 cm, a frame rate of 25 Hz, and a dynamic range of 55-65 dB. Retrospective capture is recommended, as children can abruptly move.
   NOTE: To assist with standardization of the measurement across participants and examiners and to increase efficiency in testing, the use of a preset is recommended. Most ultrasound machines have this functionality. The above settings are recommended and may vary according to the machine being used; the effects of ultrasound settings are discussed below.
6. To increase reproducibility, acquire a minimum of three insonation angles per side. Use an instrument like Meijer's Carotid Arc to standardize the angles. The angles collected here are left 210°, 240°, and 270° and right 150°, 120°, and 90°, which correspond to the anterior, lateral, and posterior views of each vessel¹⁹.
   NOTE: Collecting multiple angles is recommended as thickening is typically eccentric; however, it will increase the examination and analysis burden.
7. Ask the participant to extend their neck and tilt their head to the left at an angle of approximately 45°; a rolled towel or pillow can be wedged underneath the participant's head for comfort and to help maintain the lateral rotation¹⁹.
8. Position the transducer in the transverse scanning plane at the base of the neck with the indicator at 9 o'clock and scan upwards towards the head. Ensure the position of the indicator matches what is on the screen. Identify the common carotid artery, a pulsatile anechoic circle in the center of the screen. The jugular vein can also be seen directly atop the common carotid; it has a thinner wall and is collapsible with moderate pressure, while the carotid maintains its circular shape (Figure 1).
9. While moving up the neck, observe the common carotid artery enlarge and then bifurcate into the internal and external carotid arteries. Position the transducer at the point of enlargement, also referred to as the bulb, and turn clockwise into the longitudinal view. Ensure the indicator position is now facing towards the head.
10. Adjust gain settings to obtain symmetrical brightness for the near- and far-wall (wall closest to and wall furthest from the ultrasound beam, respectively; see Figure 2 and Figure 3) and minimal intraluminal artifact.
    NOTE: Imaging artifacts from inappropriate gain settings, such as washed-out borders, can affect the interpretation of the IMT. The IMT has a distinct double-line pattern: the intima and adventitia are hyperechoic (bright), while the media is hypoechoic (darker).
11. Obtain a digital loop with a minimum of three cardiac cycles of the common carotid artery 10 mm proximal to the bulb (Figure 2) . For optimal imaging, ensure the vessel is perpendicular to the probe beam. This can be achieved by subtly tilting the transducer on its short axis and differential pressure along the long axis of the transducer, also known as rocking or the heel-toe movement^16,17.
12. Repeat the process for the remaining two angles and adequately label each digital loop to allow for easy re-identification of the angle.
13. Repeat steps 1.7 - 1.11 on the right side of the neck. Conclude the exam. Clean off any remaining gel and remove ECG stickers. Considerations for removing ECG stickers in newborns and infants are discussed in Supplementary Table 1.

2. Aortic intima-media thickness

1. Ask the participant to lie down on the bed in a supine position with the abdomen exposed. Ask the participants to bend their knees with their feet flat on the bed; this relaxes the abdominal muscles and can improve imaging.
2. Acquire an ECG simultaneously by following step 1.4. Place the machine adjacent to the participant and within easy reach of the examiner's scanning hand. Place gel on the transducer.
   NOTE: Neonates and infants have a high breathing rate, which can strongly influence the interpretation of vessel diameter during the cardiac cycle. Hence, an ECG is essential to measure IMT at end-diastole.
3. Use a linear transducer with a minimum frequency of 7 MHz, depth to be adjusted to keep the aorta in view, a frame rate of 25 Hz, and a dynamic range of 55-65 dB. Use an appropriate zoom to maintain the aorta in the center of the screen. Use lower frequencies for those with higher body mass. Retrospective capture is recommended as per step 1.5.
4. Identify the aorta by placing the transducer in the transverse plane directly below the xiphisternum with the indicator in the 9 o'clock position (facing the sonographer). The aorta will appear as a pulsatile anechoic circle to the right of the screen. Surrounding structures include the inferior vena cava (IVC) to the left of the aorta, the liver directly above, and the anechoic vertebral body directly below.
5. Turn the probe clockwise until the aorta appears in the longitudinal view. Ensure the indicator position is facing towards the head and matches what is on the screen. In the longitudinal view, surrounding structures will include the liver and pancreas directly above and the vertebrae at the bottom of the screen.
6. Slowly move the probe downwards until the coeliac artery (CA) and superior mesenteric artery (SMA) branches are identified. Caudal to this branching is the proximal abdominal aorta, and distal to this branching is the mid to distal abdominal aorta.
7. Continue scanning until a straight segment without branching is identified. The aorta becomes more anterior as it moves distally, so adjust the zoom settings to ensure the aorta is centered on the screen. Ensure the vessel is perpendicular to the ultrasound beam and adjust gain settings as needed (Figure 5).
8. Obtain a digital loop with a minimum of three cardiac cycles. Obtain digital loops of different straight non-branching segments scanning from the proximal to the distal abdominal aorta (just before the left and right iliac arteries) and multiples for each segment. These can be labeled as proximal, middle, and distal aIMT.
   NOTE: Digital loops of different segments will not always be possible. Gas in the bowel can commonly obstruct image acquisition and limit the ultrasonic window to just proximal to the CA and SMA branching.
9. Conclude the exam. Clean off any remaining gel and remove ECG stickers.

3. Off-line intima-media analysis using semi-automated edge-detection software

1. Using a semi-automated edge detection software can lower inter-operator variability and increase reproducibility. This analysis is offline; thus, export the images in native Digital Imaging and Communications in Medicine (DICOM) format without digital compression.
2. Keep the analysis blinded to reduce potential operator bias. Practically, if there are more than two research staff members, the researcher conducting off-line image analysis should be different from the researcher who collected the images. If there is only one researcher, blinding could be achieved through deferred analysis of deidentified images.
3. Carotid IMT
   1. Select a minimum 10 mm region of interest (ROI) proximal to the bulb. The bulb can be defined as the point where the two parallel walls of the common carotid start to diverge; this is not always symmetrical and heterogeneous across individuals.
      NOTE: The recommended distance between the ROI and the bifurcation varies between children and adults and is the primary difference between them. In children, the ROI should ideally be a 10 mm region just proximal to the bulb. This is different from the recommendation for the adult population, which suggests measuring the IMT from a 10 mm straight segment at least 5 mm below the bulb^19,20.
   2. The software used automatically detects the intima-lumen and media-lumen borders for both the near and far walls. To do this, train the software on an operator-selected frame with clearly defined IMT and ask the operator to adjust the software's border detection if needed.
   3. Analyze the digital loop and report the vessel diameter, average, maximum, and minimum near- and far-wall IMT for each frame. The software also produces a trace of vessel diameter over time, which can be used to identify end-diastolic frames in the absence of an ECG (Figure 6).
      NOTE: Most semi-automated software relies on similar calculations of the IMT values. Within an ROI, multiple paired data points are drawn between the intima and media borders. Mean IMT is the average value of all points within an ROI. Maximum and minimum IMT are the single largest and smallest data points in the ROI, respectively. Due to the focal nature of plaque formation, the average maximum IMT may better indicate subclinical atherosclerosis compared to the average mean IMT³. Ideally, both methods of segmental thickness should be reported.
   4. Select diameter and far-wall IMT measurements from three consecutive cardiac cycles at end-diastole (on or near the R-wave of the ECG) and calculate the average. As the selection of end-diastolic frames is operator-dependent, record details such as loop and frame number to facilitate cross-checking of work. We recommend minimum operator intervention. Report the mean of each vessel separately, as IMT in the left common carotid can be higher than the right¹⁸.
      NOTE: Manual border adjustment is possible for difficult scans but should only be done for the selected end-diastolic frames to avoid increasing the analysis burden. If collecting the recommended three angles per side, three measurements per side will be recorded, which can be further averaged to produce overall left and right IMT.
4. Abdominal aortic IMT
   1. Select a minimum 5 mm region in a straight, non-branching segment of the vessel. Apply steps 3.2.2-3.2.3. Select diameter and far-wall IMT measurements from three consecutive cardiac cycles at end-diastole and average . Repeat if multiple digital loops have been collected and average results from all ROIs.
5. Include an expert to assess the reliability and reproducibility of both image acquisition and off-line analysis for all imaging studies. Report the outcomes of these in publications. We recommend repeats of 10% of scans. The recommended acceptable intra and inter-observer coefficient of variation is less than 6 %, or the mean difference in raw IMT measurements should be less than 0.055 mm²⁰.

Results

In this section, we represent results from prior studies to highlight key aspects of cIMT and aIMT measurement. Figure 1 and Figure 2 focus on cIMT, demonstrating both transverse and longitudinal views in young, healthy subjects and a detailed visualization of the IMT complex. Figure 3 and Figure 6 further emphasize best practices based on the positioning of the bulb, image settings, as well a...

Discussion

The present manuscript provides guidance on the acquisition and analysis of ultrasound images to measure aIMT and cIMT, specifically in younger populations (ages 0-18). Both techniques have demonstrated utility in exploring the influence of early life exposures on atherosclerosis but are susceptible to technical challenges, which we discuss below.

Critical steps in protocol implementation
Ultrasound system and settings: Acquisition of high-quality B-mode...

Materials

Name	Company	Catalog Number	Comments
12-3 MHz Broadband linear array transducer	Phillips	L12-3
Meijer's Carotid Arc	Meijer	-
Semi-automated edge detection analysis software	Medical Imaging Applications	Carotid Analyzer 5
Ultrasound	Phillips	Epiq 7
Ultrasound transmission gel	Parker	01-08