Ultrasound Assessment of Endothelial Function: A Technical Guideline of the Flow-mediated Dilation Test

Summary

The flow mediated dilation (FMD) test is the most commonly utilized, non-invasive, ultrasound assessment of endothelial function in humans. Although the FMD test has been related with the prediction of future cardiovascular disease and events, it is a physiological assessment with many inherent confounding factors that need to be considered.

Abstract

Cardiovascular disease is the primary cause of mortality and a major cause of disability worldwide. The dysfunction of the vascular endothelium is a pathological condition characterized mainly by a disruption in the balance between vasodilator and vasoconstrictor substances and is proposed to play an important role in the development of atherosclerotic cardiovascular disease. Therefore, a precise evaluation of endothelial function in humans represents an important tool that could help better understand the etiology of multiple cardio-centric pathologies.

Over the past twenty-five years, many methodological approaches have been developed to provide an assessment of endothelial function in humans. Introduced in 1989, the FMD test incorporates a forearm occlusion and subsequent reactive hyperemia that promotes nitric oxide production and vasodilation of the brachial artery. The FMD test is now the most widely utilized, non-invasive, ultrasonic assessment of endothelial function in humans and has been associated with future cardiovascular events.

Although the FMD test could have clinical utility, it is a physiological assessment that has inherited several confounding factors that need to be considered. This article describes a standardized protocol for determining FMD including the recommended methodology to help minimize the physiological and technical issues and improve the precision and reproducibility of the assessment.

Introduction

Cardiovascular disease is the leading cause of morbidity and mortality worldwide. Dysfunction of the vascular endothelium represents an initial phase toward the development of multiple vascular-related diseases¹. Hence, an accurate assessment of endothelial function in humans represents an important technique that could help in understanding the etiology of multiple cardiovascular pathologies, with the ultimate goal of improving the efficacy of the treatment and prevention of disease.

Endothelium

The endothelium is a monolayer of cells that synthesizes numerous vasoactive substances, such as nitric oxide (NO), prostacyclins, endothelins, endothelial cell growth factor, interleukins, and plasminogen inhibitors². Such factors contribute to the endothelium's function to regulate blood fluidity, vascular tone, platelet aggregation, permeability of plasma components and vessel wall inflammation^2-4. Additionally, NO plays a key anti-atherogenic role in promoting vasodilation and maintaining endothelial integrity. NO regulates vessel tone and diameter through controlling the equilibrium between the delivery of oxygen to the tissues and their metabolic demand^3,5. There are multiple endogenous, exogenous, and mechanical stimulator factors that induce endothelial NO synthase (eNOS) which synthesizes NO from L-arginine^6,7. The most notable mechanical stimulus is shear stress. Wall shear stress contributes to greater activation of eNOS, resulting in NO production and subsequent smooth muscle relaxation⁴. For that reason the decrease in NO bioavailability is often used as a measure of endothelial dysfunction⁸.

Endothelium dysfunction

The imbalance between vasodilator and vasoconstrictor factors leads to a dysfunctional endothelium². In addition, the release of inflammatory mediators and altered local shear forces may enhance the synthesis of endothelial derived reactive oxygen species (ROS). This upregulation in redox signaling not only modifies the integrity of the endothelium and reduces the synthesis of NO⁹, it can uncouple eNOS resulting in direct production of additional free radicals. Ultimately, this amelioration in NO bioavailability promotes vasoconstriction, vascular stiffness, and reduced arterial distensibility⁴.

The degree of dysfunction of the endothelium has been related with the severity of several pathologies such as hypertension¹⁰, atherosclerosis¹¹, ischemic stroke¹², diabetes¹³, preeclampsia¹⁴ or kidney diseases¹⁵ among others. Hence, there is vast interest to not only evaluate changes in endothelial function over time, but also following therapeutic interventions. Different methods have been used for the clinical assessment of endothelial function both invasively (cardiac catheterization and venous occlusion plethysmography^3,16) and non-invasively (flow mediated dilation, radial artery tonometry and pulse contour analysis^4,17,18) in coronary and peripheral circulations¹⁹.

Flow-mediated dilation

Flow mediated dilation (FMD) is a non-invasive, ultrasonic evaluation of endothelial function and has been correlated with the development of vascular health problems. Since its inception in 1989²⁰, FMD has been widely utilized as a reliable, in vivo method to evaluate predominately NO-mediated endothelial function in humans^19,21,22. Indeed, the brachial artery FMD test has been associated with other invasive techniques²³ and numerous investigations have described a strong inverse relationship between FMD and cardiovascular injury^24,25 such that individuals with more vascular pathology exhibit a lower FMD²⁵. Accordingly, these data emphasize the prognostic information that this technique can provide as it relates to future cardiovascular disease in asymptomatic subjects^26-30.

During the FMD test, the diameters of the brachial artery are continuously measured at baseline and after the release of a circulatory arrest of the forearm. Upon cuff release, the induced-reactive hyperemia promotes an increase in shear stress mediated NO release and subsequent vasodilation^19,31. FMD is expressed as the percent increase in arterial diameter following the release of the cuff compared with the diameter at baseline (FMD%).

Despite the increasing clinical interest in this technique, the FMD test is a physiological assessment and therefore, several variables need to be considered in order to conduct a precise assessment of endothelial function in humans. This article describes a standardized protocol and the recommended methodology to minimize the technical and biological issues to help improve the accuracy, reproducibility and interpretation of the FMD test.

Protocol

NOTE: The following FMD procedure is routinely conducted during vascular assessment studies in the Laboratory of Integrative Vascular and Exercise Physiology (LIVEP). All procedures followed the principles of the Declaration of Helsinki and were approved by the Institutional Review Board at Georgia Regents University. All participants were informed of the objectives and possible risks of the technique before written consent for participation was obtained. Figure 1 illustrates a schematic summary of the essential elements that should be considered for the ultrasound assessment of brachial artery FMD.

1. Subject Preparation (Prior to Arrival)

1. Confirm that the participant has abstained from practicing exercise (≥12 hr), caffeine (≥12 hr), smoking or smoke exposure (≥12 hr), vitamin supplementation (>72 hr) and any medication (≥4 hr half-lives of the drug, non-steroidal anti-inflammatory agents for 1 day and aspirins for 3 days).
2. Ensure that the participant is under fasting conditions or has only consumed low-fat meals⁴ prior to testing.
3. When testing premenopausal women, it is suggested to conduct the FMD protocol during the menses phase of the menstrual cycle to limit the impact of endogenous estrogens and progesterones^8,32,33.

2. Subject Preparation (Upon Arrival)

1. Prior to measurement acquisition, verify that the subject is resting in a supine position in a quiet, temperature-controlled (22 °C to 24 °C) room for approximately 20 min to achieve a hemodynamic steady state.
2. Attach a 3-lead ECG in the standard limb lead II position. Using American standard instrumentation, place the white/negative polarity lead just below the clavicle on the right shoulder. Connect the black/ dual polarity lead below the left clavicle near the shoulder and connect the red/positive polarity lead below the left pectoral muscle in the lateral base of the chest.
3. Extend the subject's arm laterally at about 80° of shoulder abduction and secure the distal forearm in a vacuum packed pillow to maintain accurate position of the arm during the measurement (Figure 2).
4. Place the forearm cuff immediately distal to the medial epicondyle and ensure that nothing is touching the cuff, including the table below (Figure 2).

3. Baseline Measurements

1. Mapping the Brachial Artery with the Ultrasound:
   1. While holding the probe with the hand, position it cross-sectionally and start scanning the inner side of the upper arm beginning at the insertion of the bicep and proceeding proximally.
   2. Within B-mode (gray-scale), identify the brachial artery and collateral vessels and use color flow (CF) mode to help confirm the location of the artery. Interpret the color and pulsatility carefully considering the direction of the transducer to ensure assessment of the artery and not the vein.
      NOTE: With the probe indicator facing the head, red color means flow toward the transducer (arterial flow), while blue means flow away (venous flow).
2. Identification of Brachial Artery:
   1. After finding the brachial artery, rotate the probe 90° to scan the arm longitudinally. Obtain the image between 2 to 10 cm above the antecubital fossa.
   2. Identify anatomical landmarks such as veins and fascial planes for multiple assessments in the same subject (Figure 3).
3. Securing the Probe:
   1. Secure the probe in the stereotactic probe holder. Confirm the probe is appropriately fixed to avoid excessive movements. With the probe secured in the holder, ensure that the image is as good as the image that was obtained manually without the holder.
4. Optimizing the Resolution of the Image:
   1. Optimize the image using the time gain controls (TGC's) with the probe secured.
      NOTE: An optimal image is achieved when the clearest B-mode image from the anterior and posterior intimal interfaces between the lumen and vessel wall is obtained.
   2. Have the technician manually adjust the gain, focal points, dynamic range, and harmonics to get a clear and defined image of the near and far walls of the endothelium.
5. Duplex Doppler Mode:
   1. Following B-mode acquisition, proceed to duplex scanning in the pulsed Doppler mode.
   2. Use a heel to toe approach with the probe inside the holder by rocking the transducer up on one end more than the other to adjust the brachial artery image and obtain an angle of insonation of 60°.
6. Baseline Acquisition:
   1. Obtain a satisfactory B-mode image that identifies the endothelial layers with clear intima-intima walls of the artery. Ensure that the Doppler signal appears sharp and clear sound with no muffles.
   2. Reset the ultrasound CINE loop by freezing and unfreezing the image. Press F1 to begin recording data on image software. Record baseline data for at least 30 sec. Analyze the average diameter and blood velocity for 30 sec to represent baseline values. Note: Different ultrasounds and software set-ups may require different sequences to obtain the required action.

4. Vascular Occlusion Measurements

1. Forearm Occlusion:
   1. Rapidly inflate the forearm occlusion cuff, using compressed air, to supra-systolic pressures (250 mm Hg) for 5 min to induce arterial occlusion.
   2. After 4 min and 30 sec of forearm occlusion, begin acquiring data.
      NOTE: Occlusion measurements will be represented by the last 30 sec of occlusion.

5. Reactive Hyperemia (Post Cuff Release) Measurements

1. Continuing to Acquire Data from Pre-cuff Release:
   1. Deflate the cuff at 5 min.
   2. Maintain recording for two minutes following cuff release.
2. Following 2 min of post cuff release recording, stop and save the recordings. The highest 5 sec averaged interval throughout the 2 min post-occlusion collection period will be used to represent the peak hyperemic diameter.

6. Analysis of the Results: Edge Detection and Wall Tracking

1. Due to the complexity of FMD analysis, use edge-detection and wall-tracking software throughout FMD testing for higher reproducibility according to manufacturer's instructions.
   NOTE: This offline analysis is less operator dependent than the manual assessment and therefore improves the accuracy of the FMD data^4,34-36. In addition, this off-line analysis system also permits the synchronization with the ECG for the identification of end-diastolic arterial diameters, avoiding distortion of pulse-related changes in diameter⁴. It should be noted that, although the use of ECG is endorsed to minimize pulsation variability, it is also possible to perform the FMD protocol without ECG gaiting³⁷. Although not recommended, if edge computer-assisted analysis is unavailable, careful manual assessment of both diameter and velocities should be collected³⁶.
2. For the evaluation of vessel diameters, it is necessary to visually inspect each frame to determine the best placement of the ultrasonic calipers along the B-mode image³⁸.
   NOTE: Regardless of data analysis method, it is recommended to collect diameter and velocity data every 4 sec during the first 20 sec of reactive hyperemia and every 5 sec for the remaining post occlusion period⁴.

Results

Baseline characteristics from an apparently healthy cohort group are presented in Table 1. The most common variables of FMD testing conducted in the Laboratory of Integrative Vascular and Exercise Physiology (LIVEP) are presented in Table 2. The following variables are considered the main FMD parameters to analyze by the published FMD tutorial⁴ and guidelines³⁶.

Baseline an...

Discussion

Introduced in 1989²⁰, the FMD test has been widely used in humans as a non-invasive measure of endothelial function. FMD has not only been shown to predict future vascular-related disease risk^19,52,53, lower FMD values have been show to strongly correlate with cardiovascular impairments^24,25,54. Although there are other techniques to assess endothelial function, both invasively (coronary angiography) and non-invasively (venous plethysmography and finger plethysmography), FMD has been the ...

Materials

Name	Company	Catalog Number	Comments
Doppler ultrasound	GE Medical Systems	Logiq 7	Essential to include Duplex mode for simultaneous acquisition of B-mode and Doppler
Electrocardiographic (ECG) gating	Accusync Medical Research	Accusync 72
12-MHz Linear array transducer	GE Medical Systems	11L-D	A high-resolution linear array probe is essential
Forearm occlusion cuff	D.E. Hokanson	SC5	5 cm x 84 cm
Ultrasound transmission gel	Parker	01-08
Rapid cuff inflator	D.E. Hokanson	E-20 AG101
Sterotactic-probe holder	Flexabar	18047	Magnetic base fine adjustor
Edge detection analysis software	Medical Imaging Applications	Brachial Analyzer 5