An Image-Free Ultrasound-Based Approach for Combined Assessment of Local and Regional Arterial Stiffness in Humans

Summary

This protocol describes an image-free ultrasound-based technique for simultaneously assessing both local and regional arterial stiffness in humans. The technique employs a single-element piezoelectric ultrasound probe to insonate the common carotid artery and an oscillometric cuff to capture the femoral pulse waveform to determine local and regional arterial stiffness.

Abstract

Arterial stiffness is a cardinal marker of vascular aging, representing the cumulative impact of mechanical and biological stressors on the vessel wall resulting from aging and various cardiovascular risk factors, which lead to alterations in the material properties and wall mechanics of both central and peripheral arteries. Arterial stiffness has been identified as an independent predictor of cardiovascular morbidity and mortality. Based on whether the stiffness assessment is performed at a specific anatomical location or across a segment of the arterial tree, arterial stiffness measurements are broadly categorized as local and regional assessments. Local measurement involves quantifying arterial wall properties, including compliance, distensibility, or elastic modulus, while regional assessment is based on measuring the velocity of pulse wave propagation along an arterial segment. Here, we present a validated, image-free, ultrasound-based approach capable of simultaneously assessing both local and regional arterial stiffness in humans. The technique involves insonating the common carotid artery using a single-element piezoelectric ultrasound probe and real-time tracking of echoes originating from the arterial walls to capture the carotid artery distension waveform. Local measures of arterial stiffness are derived from distension data and carotid blood pressure. An oscillometric cuff applied to the thigh is used to simultaneously acquire the femoral pulse waveform and compute carotid-femoral pulse wave velocity by determining pulse travel times to the carotid and femoral sites, along with surface-measured arterial path lengths.

Introduction

Arterial stiffness is a key marker of vascular ageing, reflecting the cumulative impact of mechanical and biological insults associated with ageing and various cardiovascular risk factors on the arterial wall^1,2. Pathobiologically, these changes involve elastin degradation, collagen deposition and cross-linking, smooth muscle stiffening, and alterations in the extracellular matrix of the arterial wall, making it stiffer and less compliant, thereby compromising the Windkessel functions of large elastic arteries^1,2.

Physiological consequences of the large artery stiffening (LAS) are multi-faceted. An increase in the characteristic impedance of the aorta and the systolic afterload acting on the left ventricle disturbs normal ventricular-arterial interactions and promotes myocardial dysfunction, myocardial fibrosis, and left ventricular hypertrophy^1,2. Additionally, the loss of the mechanical buffering attributed to the Winkessel function amplifies pressure and flow pulsatility in the microvasculature of various target organs, including the brain and kidney, leading to microvascular and parenchymal dysfunction in these vital organs^1,2. These pathophysiological constructs of LAS and its consequences are replicated epidemiologically by large population-based studies that have irrefutably revealed the significance of arterial stiffness as an independent predictor of cardiovascular morbidity and mortality beyond conventional cardiovascular risk factors^3,4,5,6. Accumulating evidence in this respect has paved the way for the inclusion of arterial stiffness assessment in clinical practice guidelines, if a risk-based BP-lowering treatment decision remains uncertain for individuals with elevated BP⁷. These developments underscore the importance of access to valid and reliable tools for the non-invasive measurement of arterial stiffness in humans, particularly for vascular health assessment and risk stratification.

Conventional approaches to quantifying arterial stiffness are two-pronged^2,8: (1) record the pulsatile changes in arterial diameter and pressure at a specific location in the arterial tree to calculate arterial distensibility or other indices as a 'local' measure of arterial stiffness, (2) measure the velocity of propagation of the arterial pulse between two points in the arterial tree to determine the pulse wave velocity (PWV) as a regional measure of arterial stiffness. Theoretically, PWV is inversely related to the square root of distensibility, as established by the Bramwell-Hill equation, which demonstrates the physiological concurrence between the two broader measures of arterial stiffness⁸. Quantifying PWV of large central arteries involves the use of dedicated sensors, including tonometers, to non-invasively record the arterial pulse waveforms from two accessible locations in the arterial tree. Carotid-Femoral PWV, the gold standard measure of large artery stiffness, is calculated using sequentially or simultaneously captured arterial pulse waveforms from the common carotid and femoral arteries^2,8.

Quantifying local measures of arterial stiffness often involves the use of imaging modalities, including ultrasound or MRI, and requires exhaustive post-processing of the image sequences to extract diameter and area data for the calculation of stiffness indices^8,9. Measures of local arterial stiffness, when captured from the barosensitive locations of the arterial tree, can also provide vital physiological insights into the mechanical gain of the baroreceptor reflex arc, a pathway crucial for short-term cardiovascular homeostasis, which is often deranged in neurological and metabolic disorders^10,11. In addition to stiffness indices, ultrasound-based Doppler and vector flow imaging techniques provide quantitative measures of blood flow velocity, flow rate, and wall shear stress, which help characterise local hemodynamic environments relevant to atherosclerosis and vascular remodelling^12,13. Similarly, phase-contrast and 4D flow MRI yield volumetric blood flow, pressure gradients, wall shear stress, turbulent flow patterns, vorticity, and aortic pulse wave velocity, offering a comprehensive, non-invasive characterisation of hemodynamics that complements arterial wall stiffness measurements^14,15. Recently, cuffless blood pressure (BP) technologies based on photoplethysmography (PPG) have been developed to estimate local or regional BP and pulse wave velocity from peripheral pulse waveforms^16,17. These systems typically derive BP from PPG-based pulse transit time or pulse wave analysis, sometimes in combination with ECG or multi-site PPG, and can track beat-to-beat changes in local arterial stiffness and hemodynamics in a wearable or smartphone-based form factor^16,17,18.

We present here a validated, image-free ultrasound-based technique for simultaneously assessing both local and regional arterial stiffness in humans^9,19,20. The method employs a single-element piezoelectric ultrasound probe to insonate the common carotid artery, capturing real-time echo signals from the arterial walls to generate the carotid artery distension waveform. Local arterial stiffness is quantified using the distension data in conjunction with arterial blood pressure. To evaluate regional arterial stiffness, an oscillometric cuff is applied to the thigh to capture the femoral pulse waveform non-invasively. Carotid-femoral pulse wave velocity (cf-PWV) is then calculated by measuring the pulse arrival delay between the femoral and carotid sites, along with surface-measured arterial path lengths.

Protocol

The study protocols were approved by the Institute Ethics Committee, All India Institute of Medical Sciences, New Delhi (IEC-477/17.06.2022, RP-04/2022). Written informed consent was obtained from all the participants who volunteered for the study. The consumables and equipment used are listed in the Table of Materials.

1. Equipment components and connections

NOTE: The measurement setup consists of the ARTSENS Plus PWV device (image-free arterial stiffness measurement device), two inflatable cuffs (brachial and femoral), a hosepipe, an A-mode ultrasound transducer, a micro-USB cable, measuring tape, and a charging adapter. The interfaces available on the device are shown in Figure 1. The following steps must be followed to prepare the device for measurements:

1. Connect the power adapter to the device's power port and plug the other end into an AC power outlet. This step may be omitted if the device has an adequate battery backup.
2. Ensure the host computer is powered during operation to avoid any interruption in processing or communication.
3. Connect the hosepipe to the cuff port on the image-free arterial stiffness measurement device until a "click" sound confirms secure locking.
4. Attach the cuff (either brachial or femoral) to the other end of the hosepipe, depending on the desired measurement. The connector uses a twist-to-lock mechanism.
5. Connect the micro-USB cable to the device and the other end to the host computer. A direct connection is recommended. If the host does not have USB Type-A ports, use a powered USB 2.0 hub to ensure stable data transfer.

2. Assessment preparation

NOTE: To begin a new measurement, launch the dedicated software application to run the image-free arterial stiffness measurement device (software build version: V1.0) on the host computer and follow these steps:

1. Select NEW PATIENT from the Home page to initiate a new test session.
2. Enter the patient information and proceed to the Assessment Preparation page by clicking on Right Arrow button in the bottom right of the page. If any mandatory fields are incomplete or contain invalid entries, the software will display an error message at the bottom of the screen.
3. Instruct the subject to rest in a supine position for at least 5 min prior to the measurement.
4. Place the brachial and femoral cuffs on the left arm and left thigh, respectively, ensuring they are snug but not excessively tight.
5. Connect the hosepipe to the brachial cuff. Ensure the connection is secure with no air leakage. An error message will appear during the subsequent steps if leakage or improper cuff placement is detected.
6. Click on Measure Values to automatically record systolic, diastolic, and mean arterial pressures, along with heart rate.
7. Enter anthropometric data if available.
8. Palpate the left side of the neck to locate the carotid pulse.
9. Measure and enter (in millimeters) the following arterial path lengths (as shown in Figure 2):
   1. Distance from the carotid artery site to the sternal notch
   2. Distance from the sternal notch to the top of the thigh cuff
   3. Distance from the femoral artery site to the top of the thigh cuff
      NOTE: These distances are illustrated in the software interface to assist accurate entry. The distance should not use patient contact where the individual contours of the patients' body can incorrectly increase PWV distance based on their own individual anatomical external body structure. Also, multiply measurements in centimeters by 10 to convert them to millimeters and enter the distances into the software.
10. Disconnect the brachial cuff from the hosepipe and connect it to the thigh cuff.
11. Navigate to the next screen to begin vascular stiffness measurements by clicking on the Right Arrow button at the bottom right of the page. The software verifies probe and cuff connectivity. The cuff will inflate and deflate as part of an automatic check before proceeding to the Measurement screen.

3. Vascular stiffness measurement

1. Apply a drop of ultrasound gel to the A-mode transducer.
2. Position the probe over the location where carotid pulsations are felt (refer to Figure 3).
3. Ensure that the measurement screen displays the following feedback:
   1. RF waveform: Ensure that it displays the raw ultrasound echo signal from the carotid artery ( Figure 4, right column).
   2. Signal quality bars: Indicate the quality of echo signals for the near wall (NW) and far wall (FW) of the artery. These are situated right below the RF waveform.
   3. Progress bar: Ensure that it indicates progress as valid cardiac cycles are recorded.
   4. Pulse waveforms: Show carotid distension and femoral pressure pulse waveforms.
4. Move and orient the probe to locate the carotid artery (refer to Figure 4) using the RF waveform display.
   NOTE: Proper placement is confirmed when two vertically aligned echo patterns (corresponding to the artery's near and far walls) exhibit opposite-phase motion.
5. Ensure that the artery detection is visually indicated by a pair of cursors bounding the artery walls.
   NOTE: The femoral cuff inflates automatically during this process. A change in waveform color from red to blue indicates adequate cuff inflation. Once the probe is stably held, both carotid and femoral waveforms appear. These signals are quasi-periodic in nature.
6. Record the waveforms for a user-defined duration, which is indicated by the filling progress bar. The default setting requires at least 10 high-quality cardiac cycles from both the carotid and femoral arteries.
   NOTE: Upon successful acquisition of valid data, the progress bar reaches its maximum, and the Results page is displayed. The number of cycles required for results, and consequently the recording duration, is configurable by the operator.

4. Obtaining device outputs

1. Check the list of direct measurements:
   1. Brachial blood pressure (SBP, DBP, MAP) by oscillometry.
   2. Carotid diameter pulse waveform and absolute values (systolic, diastolic, peak distension) by ultrasound.
   3. Femoral pressure pulse waveform by cuff transducer.
   4. Carotid-femoral pulse transit time (cf-PTT) measured directly from the time delay between the acquired carotid and femoral waveforms, without conversion to an estimated pressure waveform.
2. Check the list of derived measurements:
   1. Central blood pressure (PS, PD) derived from brachial blood pressure and carotid diameter waveforms. (It is obtained by calibrating the diameter waveform using a fundamental nonlinear pressure-diameter relationship, under the assumption of consistent end-diastolic and mean pressures across the arterial tree^21,22.)
   2. Carotid stiffness indices: stiffness index (β), elastic modulus (Ep), and arterial compliance (AC) evaluated using the direct measurements as shown in Table 1.
   3. Carotid-femoral pulse wave velocity (cf-PWV), the gold-standard regional stiffness metric evaluated using the direct measurements as shown in Table 1.

5. Configuration of the device for reliable measurements

1. Measurement settings:
   1. Set the transducer frequency to 10 MHz.
      NOTE: A 5 MHz transducer is also supported and can be selected in the basic settings.
   2. Set the ultrasound signal sampling rate to 80 MHz.
   3. Set the ultrasound frame rate to 250 Hz.
   4. Set the femoral pressure pulse-wave sampling rate to 250 Hz.
   5. Apply a Golay filter with 20 side-points for signal smoothing.
   6. Set the cuff inflation pressure set-point to 10 mmHg below the patient's DBP.
2. Probe handling:
   1. Refer to Figure 3 to identify the ideal region for palpation and transducer placement.
   2. Palpate the indicated area to locate the strongest carotid pulse to ensure optimal signal quality.
   3. Apply gentle and consistent pressure to maintain probe contact. Avoid applying excess pressure, as this can distort the signal and cause patient discomfort.
   4. If necessary, rest the operator's wrist on a support surface to improve stability.
3. Cuff placement:
   1. Wrap the cuff around the mid-thigh, ensuring the hosepipe exits downward.
   2. Position the Velcro wraps over the inner thigh.
   3. Ensure the cuff is snug by holding one end firmly on top of the thigh and wrapping clockwise with the other hand.
   4. Verify that the hosepipe is not trapped beneath the cuff fabric.

6. Using the software GUI for real-time measurement feedback

1. Observe the RF waveform display. Initially unstructured, two echo traces with opposite motion will appear in the upper third of the display once the artery is located.
2. Monitor the signal quality bars adjacent to the RF waveform. Aim for a fill level above 50% (indicated by the bars turning from white to green).
3. Use the displayed probe status, cuff status, femoral pressure waveform color, and other system diagnostics to guide the acquisition.
4. Ensure that the host computer is properly configured.
5. Keep the host computer connected to a power outlet throughout the entire measurement procedure to prevent performance throttling that can adversely affect data acquisition and USB communication.

Results

Figure 4 presents representative ultrasound recordings, illustrating both good- and poor-quality signals resulting from correct and incorrect probe orientation, respectively. A sample results page is shown in Figure 5, indicating the measured stiffness and blood pressure metrics. (Note that the page is loaded with results for illustration purposes). For individual stiffness measurement, local and regional, the age-associated reference ranges are adopted from the...

Discussion

The method described enables the simultaneous assessment of both local and regional arterial stiffness in humans using a single, image-free, ultrasound-based examination. This approach integrates a single-element piezoelectric ultrasound probe to capture carotid artery diameter waveforms for local stiffness assessment and a thigh cuff to record the femoral pulse waveform for determining carotid-femoral pulse wave velocity (cf-PWV). This dual measurement approach provides a comprehensive assessment of arterial health with...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ARTSENS Plus unit	VascRisk LLC	AP020201001	Dimensions : 240 mm X 170 mm x 60 mm, Weight: ~1Kg
AC power adaptor	VascRisk LLC	AP020201005	2 MOPP Class II, 9V - 3.3A, 36W
USB cable	VascRisk LLC	AP020201009	1 x Micro USB to USB type A cable; Length: 1m
Thigh cuff	VascRisk LLC	AP020201002	Adult and Standard size
Brachial cuff	VascRisk LLC	AP0203020021	Adult and Standard size
Hosepipe	VascRisk LLC	AP020201004	1 x 1/8" pipe; Length: 1.5m
Ultrasound transducer probe	VascRisk LLC	AP020201008	1 x Single element Piezo ultrasonic Probe
Measuring tape	VascRisk LLC	AP020201010	1 x 150cm, with 1mm resolution
Host computer	User's preferance	Minimum requirements: Intel i5 Processor, 8GB RAM, Microsoft Windows 10 and above, 1 x USB2.0 port
Software drive	VascRisk LLC	AP020202056	ISO installation file; < 1 GB