Technique for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery
Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery
Journal of the American College of Cardiology Volume 39, Issue 2, 16 January 2002, Pages 257-265
A report of the International Brachial Artery Reactivity Task Force
Mary C. Corretti MD, FACC, Todd J. Anderson MD, Emelia J. Benjamin MD, MSc, David Celermajer MD, Francois Charbonneau MD, Mark A. Creager MD, John Deanfield MD, Helmut Drexler MD, Marie Gerhard-Herman MD, David Herrington MD, MHS, Patrick Vallance MD, Joseph Vita MD and Robert Vogel MD
The complete document (copyright Journal of the American College of Cardiology) is publicly available by searching PubMed.
Factors affecting flow-mediated vascular reactivity:
The subject is positioned supine with the arm in a comfortable position for imaging the brachial artery. The brachial artery is imaged above the antecubital fossa in the longitudinal plane. A segment with clear anterior and posterior intimal interfaces between the lumen and vessel wall is selected for continuous 2D grayscale imaging. Currently, cross-sectional imaging of the brachial artery cannot be used to determine maximum diameter or area of the lumen because of inadequate image definition of the lateral walls. Also, skew artifacts from cross-sectional imaging limit accurate diameter determination. In addition to 2D grayscale imaging, both M mode and A mode (wall tracking) can be used to continuously measure the diameter, yet these techniques may be more subject to error owing to tracking drift. No direct comparison has been made of diameter determinations from continuous recording using grayscale images versus wall tracking. During image acquisition, anatomic landmarks such as veins and fascial planes are noted to help maintain the same image of the artery throughout the study. A stereotactic probe-holding device can be helpful.
To create a flow stimulus in the brachial artery, a sphygmomanometric (blood pressure) cuff is first placed either above the antecubital fossa or on the forearm. A baseline rest image is acquired, and blood flow is estimated by time-averaging the pulsed Doppler velocity signal obtained from a midartery sample volume. Thereafter, arterial occlusion is created by cuff inflation to suprasystolic pressure. Typically, the cuff is inflated to at least 50 mm Hg above systolic pressure to occlude arterial inflow for a standardized length of time. This causes ischemia and consequent dilation of downstream resistance vessels via autoregulatory mechanisms. Subsequent cuff deflation induces a brief high-flow state through the brachial artery (reactive hyperemia) to accommodate the dilated resistance vessels. The resulting increase in shear stress causes the brachial artery to dilate. The longitudinal image of the artery is recorded continuously from 30 s before to 2 min after cuff deflation. A midartery pulsed Doppler signal is obtained upon immediate cuff release and no later than 15 s after cuff deflation to assess hyperemic velocity.
Studies have variably used either upper arm or forearm cuff occlusion, and there is no consensus as to which technique provides more accurate or precise information. When the cuff is placed on the upper part of the arm, reactive hyperemia typically elicits a greater percent change in diameter compared with that produced by the placement of the cuff on the forearm. This may be due to a greater flow stimulus resulting from recruitment of more resistance vessels or possibly to direct effects of ischemia on the brachial artery. However, upper-arm occlusion is technically more challenging for accurate data acquisition as the image is distorted by collapse of the brachial artery and shift in soft tissue. The change in brachial artery diameter after cuff release increases as the duration of cuff inflation increases from 30 s to 5 min. The change in diameter is similar after 5 and 10 min of occlusion; therefore, the more easily tolerated 5-min occlusion is typically used. Also, FMD may be studied in the radial, axillary and superficial femoral arteries. Notable caveats are that arteries smaller than 2.5 mm in diameter are difficult to measure, and vasodilation is generally less difficult to perceive in vessels larger than 5.0 mm in diameter.
Endothelium-independent vasodilation with nitroglycerin
At least 10 min of rest is needed after reactive hyperemia (i.e., FMD) before another image is acquired to reflect the reestablished baseline conditions. In most studies to date, an exogenous NO donor, such as a single high dose (0.4 mg) of nitroglycerin (NTG) spray or sublingual tablet has been given to determine the maximum obtainable vasodilator response, and to serve as a measure of endothelium-independent vasodilation reflecting vascular smooth muscle function. Peak vasodilation occurs 3 to 4 min after NTG administration; images should be continuously recorded during this time, and NTG should not be administered to individuals with clinically significant bradycardia or hypotension. Determining the vasodilator responses to increasing doses of NTG, rather than a single dose, may further elucidate changes in smooth muscle function or arterial compliance that might be playing a role in any observed changes in FMD.
Accurate analysis of brachial artery reactivity is highly dependent on the quality of ultrasound images.
The diameter of the brachial artery should be measured from longitudinal images in which the lumen-intima interface is visualized on the near (anterior) and far (posterior) walls. These boundaries are best visualized when the angle of insonation is perpendicular. Thus, clear visualization of both the near and far wall lumen-intima boundaries indicates that the imaging plane is bisecting the vessel in the longitudinal direction, and diameters measured from these images likely reflect the true diameter. Once the image for analysis is chosen, the boundaries for diameter measurements (the lumen-intima or the media-adventitia interfaces) are identified manually with electronic calipers or automatically using edge-detection software. The variability of the diameter measurement is greatest when it is determined from a point-to-point measurement of a single frame, and least when there is an average derived from multiple diameter measurements determined along a segment of the vessel.
Similarly, cross-sectional images are less reliable, for only a single point in the vessel's length is used to determine maximal diameter. The diameter measurement along a longitudinal segment of vessel is dependent upon the alignment of the image. Skew occurs when the artery is not completely bisected by the plane of the ultrasound beam. With slight skew, the maximal diameter measured is constant, and thus yields a more accurate measurement. Some edge-detection programs can account for skew from transducer angulation.
Timing of FMD
Flow-mediated vasodilation is an endothelium-dependent process that reflects the relaxation of a conduit artery when exposed to increased shear stress. Increased flow, and thereby increased shear stress, through the brachial artery occurs during postocclusive reactive hyperemia. Several studies have suggested that the maximal increase in diameter occurs approximately 60 s after release of the occlusive cuff, or 45 to 60 s after peak reactive hyperemic blood flow. The increase in diameter at this time is prevented by the NOS inhibitor Ng-monomethyl-L-arginine, indicating that it is an endothelium-dependent process mediated by NO. Other measures of vasodilator response include time to maximum response, duration of the vasodilator response and the area under the dilation curve.
Timing of the measurement during the cardiac cycle
Brachial artery diameter should be measured at the same time in the cardiac cycle, optimally achieved using ECG gating during image acquisition. The onset of the R-wave is used to identify end diastole, and the peak of the T-wave reproducibly identifies end systole. Peak systolic diameter is larger than end systolic diameter, because the vessel expands during systole to accommodate the increase in pressure and volume generated by left ventricular contraction. The magnitude of systolic expansion is affected by the vessel compliance, and it may be reduced by factors such as aging and hypertension (possibly by reduced bioavailability of NO). Thus, functional characteristics of the brachial artery may obfuscate the measurement of FMD if diameter is measured during end systole; however, this concern has not been tested in a rigorous trial.
Flow-mediated vasodilation is typically expressed as the change in post-stimulus diameter as a percentage of the baseline diameter. Baseline diameter influences percent change in two ways. First, for any given absolute change in the postflow stimulus diameter, a larger baseline diameter yields a smaller measure of percent change. Reporting absolute change in diameter will minimize this problem. Second, smaller arteries appear to dilate relatively more than do larger arteries. Both factors merit consideration when comparing vasodilator responses between individuals and groups with different baseline diameters. For studies in which comparisons are made before and after an intervention in the same individuals, percent change might be the easiest method to use if baseline diameter remains stable over time. However, the best policy may be to measure and report baseline diameter, absolute change and percent change in diameter.
Training and quality improvement
Despite its deceptively simple appearance, ultrasonographic assessment of brachial artery reactivity is technically challenging and has a significant learning curve. Ideally, an individual trained in the principles and technical aspects of 2D and Doppler ultrasonography would perform the technique. The learning curve typically requires several months and depends both on the technical skill of the individual and the frequency with which the technique is performed. Optimal training in the technique requires hands-on training by an experienced individual who can demonstrate the pitfalls and ultrasound artifacts and who can delineate manual techniques and optimal ultrasonography system parameters.
Thorough training in the technique helps to establish high quality and consistency in the method and data. An important component of training and protocol development is attention to ergonomic issues. The operator should sit in a comfortable position and support the arm holding the probe. Both the quality of the images and the measurements rely on steady transducer imaging of the brachial artery while minimizing stress-related fatigue and injuries. It is recommended that at least 100 supervised scans and measurements be performed before independent scanning and reading is attempted; 100 scans per year should be performed to maintain competency. This recommendation is based in part on criteria for ultrasound proficiency established by the Intersocietal Commission for the Accreditation of Vascular Laboratories. Ongoing feedback from the trainer and review of videotapes showing recorded brachial artery vasoactivity testing provide valuable education. Criteria for acceptable image quality for optimal FMD measurements set a useful standard to qualify brachial artery studies for research protocols.
Evaluating precision of the technique
Intraobserver and interobserver variability in image acquisition and analysis should be established and periodically reassessed for each condition, including baseline, reactive hyperemia and NTG administration. Image variability is best judged by having two sonographers independently scan the same series of subjects at different times. The highest reproducibility is likely to be shown over a short interval, during which the individual vasodilator response is unlikely to have changed owing to environmental or other influences. This can be accomplished by taking two measurements on the same day within a 10- to 15-min interval, or on separate days in otherwise identical circumstances. Longitudinal studies in which interventions over weeks to months are tested require that reproducibility measurements be performed at longer intervals. The image analysis and measurement of the vasodilator response from repeated studies should be performed by an individual who is blinded as to sequence. Measurement variability is assessed, typically, by a designated core laboratory for multicenter trials, prior to site certification and periodically thereafter to analyze for temporal drifts.
Several approaches exist to describe the differences in any two sets of measurement results. One is the correlation coefficient, which is derived from data that represent the entire range of measurements anticipated in the setting in which the technique will be employed. A second metric is simply the mean and range of differences between the measures, which gives an intuitive understanding of the lower limits of differences that can meaningfully be ascribed to variation between subjects or secondary to intervention. The third metric, the coefficient of variation, is intended to communicate the size of the variance of a measure relative to the mean value of what is being measured. Because FMD is a percentage-ratio measure, small differences between observers appear very large.
There is no single ideal measurement to assess reproducibility of this technique. A scatterplot showing results obtained at time one and time two along with the line of identity, accompanied by the results of the three metrics described in the previous text, is likely the most complete way to describe reproducibility of FMD of the brachial artery. Rigorous attention to protocol standardization, training and ongoing quality improvement is critical to generating valid, reproducible data.
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