Tutorial

Ultrasound Assessment of Flow-Mediated Dilation

Ryan A. Harris, Steven K. Nishiyama, D. Walter Wray, Russell S. Richardson

AbstractDeveloped in 1992, the flow-mediated dilation test is now the most commonly used noninvasive assessment of
vascular endothelial function in humans. Since its inception, scientists have refined their understanding of the
physiology, analysis, and interpretation of this measurement. Recently, a significant growth of knowledge has added to
our understanding and implementation of this clinically relevant research methodology. Therefore, this tutorial provides
timely insight into recent advances and practical information related to the ultrasonic assessment of vascular endothelial
function in humans. (Hypertension. 2010;55:1075-1085.)
Key Words: FMD endothelial function reactive hyperemia shear stress

T his tutorial provides an easy-to-follow reference for

researchers interested in performing flow-mediated dila-
on 250 studies that used the measurement of FMD and
revealed that technical aspects of the measurement (ie,
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tion (FMD) testing for the first time, but also, because of the occlusion location and duration) may explain the differences
numerous recent advancements in this field, an important and in FMD observed among studies.5 At this time, Deanfield et
timely updated summary of current practices for established al6 published their recommendations for global endothelial
researchers in the area. Because vascular endothelial dysfunc- function testing with a specific section highlighting the
tion represents an initial step toward hypertension and car- noninvasive FMD technique. Most recently, Pyke and Tscha-
diovascular disease, the accurate assessment of vascular kovsky7 provided an update to the guidelines presented by
endothelial function is an essential tool that will assist in our Corretti et al4 that specifically targeted the issue of the shear
understanding of the etiology of these vascular-related dis- stress stimulus and have provided important recommenda-
eases and determine the efficacy of therapeutic treatments tions that are now common practice for FMD testing. Despite
that target vascular health. Beyond a short introduction of the these considerable advancements in the understanding and
history and physiology behind the ultrasonic assessment of application of the FMD technique, this comprehensive tuto-
FMD, there follows comprehensive, evidence-based, techni- rial offers up-to-date technical instructions for the perfor-
cal, and interpretive strategies for performing the ultrasonic mance and interpretation of FMD.
assessment of endothelial function in humans.
Endothelium
Historical Perspective The endothelium plays multiple pathological and physiolog-
In 1992, Celermajer et al1 developed the FMD technique as a ical roles, including the regulation of smooth muscle tone,
noninvasive method to measure vascular endothelial function. control of thrombosis, inhibition of leukocyte and platelet cell
Since this time, the ultrasonic assessment of FMD in response adhesion, and promotion of intra-arterial permeability.8 10 In
to occlusion-induced hyperemia has been established as a addition, there are numerous vasoactive substances released
reliable, noninvasive measurement of endothelial function2 from the endothelium, including prostacyclins, endothelins,
and has been documented to correlate with invasively as- endothelial cell growth factors, interleukins, plasminogen
sessed endothelial function in the coronary arteries.3 In an inhibitors, and NO. The latter is, perhaps, the major mediator
effort to standardize this measurement among investigators, of vasodilation11 and has, thus, been intensely studied since
in 2002 Corretti et al4 published the initial guidelines for the its discovery in 1980.12 After 30 years of NO-related
ultrasonic assessment of FMD of the brachial artery, which, research, reduced NO bioavailability has become synony-
to date, have been referenced 1000 times. Since then, an mous with the condition broadly described as endothelial
ongoing effort has been made to adapt the original method- dysfunction.13 In addition to being proposed as the primary
ology introduced by Celermajer et al,1 to a more robust etiology of atherosclerosis,14 endothelial dysfunction is the
assessment of a true NO-dependent measurement of vascular earliest identifiable event in the process of atherosclerotic
endothelial function. In 2005, a meta-analysis was conducted cardiovascular disease, the leading cause of morbidity and

Received January 22, 2010; first decision February 9, 2010; revision accepted March 5, 2010.
From the Georgia Prevention Institute (R.A.H.), Medical College of Georgia, Augusta, Ga; College of Osteopathic Medicine (S.K.N.), Touro
University, Henderson, Nev; Division of Geriatrics, Department of Medicine (D.W.W., R.S.R.) and Department of Exercise and Sport Science University
of Utah (D.W.W., R.S.R.), Salt Lake City, Utah; Geriatric Research, Education, and Clinical Center (D.W.W., R.S.R.), Salt Lake City Veterans Affairs
Medical Center, Salt Lake City, Utah.
Correspondence to Ryan A. Harris, 1120 15th St, Medical College of Georgia, Georgia Prevention Institute, Augusta, GA 30912. E-mail
ryharris@mcg.edu
2010 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org DOI: 10.1161/HYPERTENSIONAHA.110.150821

1075
 1076 Hypertension May 2010

mortality in the United States.15 It follows that the assessment Duplex Mode
of endothelial function has become an area of considerable Duplex mode allows the simultaneous acquisition of B-mode
interest to the medical and research communities. and Doppler for the determination of vessel diameter and
blood velocity, respectively. These collective measurements
Flow-Mediated Dilation enable the calculation of shear rate (see below) for any given
When measured appropriately, the assessment of endothelial time period from which the integral of shear across time (ie,
function via FMD has been proposed to represent a functional shear rate area under the curve [AUC]) may be calculated.
bioassay for endothelium-derived NO bioavailability in hu- This is a key advantage of Duplex scanning, because it is the
mans.13 During an FMD test, vasodilation occurs after an cumulative exposure to shear experienced by the artery that is
acute increase in blood flow, typically induced via circulatory proposed to be the major stimulus for the FMD response.23
arrest in the arm (suprasystolic cuff occlusion) for a period of Angle Steer and Insonation Angle Correction
time. Specifically, this hyperemia increases laminar shear With a blood vessel that runs parallel to the surface of the skin
forces parallel to the long axis of the vessel,16 which is (eg, brachial artery), the simple placement of a high-
transduced via luminal mechanoreceptors to the endothelial frequency linear ultrasound probe has the potential to yield an
cell. This event increases G-protein expression of phosphoki- excellent image, because the ultrasound beam will bisect the
nase A, signaling an increase of endothelial NO synthase vessel at 90. However, this is the worst-case scenario for the
activity, which catalyzes the conversion of L-arginine to Doppler assessment of blood velocity, because the accuracy
NO.17 NO then diffuses into the tunica media, where it of this measurement technique varies from minimal error at
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activates soluble guanylate cyclase, which converts GTP into 0 (parallel with the blood flow) to virtually no signal (0
GMP to induce relaxation of the smooth muscle and subse- velocity) when the beam bisects the artery at 90 (the
quent vasodilation. In its traditional form, the increase in optimum angle for imaging). Some compromising of image
arterial diameter, as a consequence of the reactive hyperemia, quality by rocking the transducer (probe) up on one end more
is compared with the baseline diameter and expressed simply than the other (known as heel/toe adjustments) will make the
as a percentage of this baseline diameter (% FMD). Despite vessel appear to run diagonally across the monitor and bring
this intuitive and attractive link between FMD testing the Doppler beam to an angle of 90. Even with excellent
and NO bioavailability, it should be noted that vessel type technique, to actually attain the accepted insonation angle of
60 in the brachial artery is certainly challenging if not
and size may influence the relative contribution of NO,18 and
impossible. This issue is resolved through use of angle
there is still some debate about this in the literature with data
steer, achieved by asynchronous firing of the phased array of
both for13,19,20 and against the concept that vasodilation
pizo-electric crystals that transmit and receive the Doppler
mediated by the endothelium is predominantly a consequence
signal. With the beam steered left or right 20 to 30, an
of NO.21,22
insonation angle of 60 can be achieved with far less heel/toe
movements of the probe and sacrifice of image quality.
Measurement of FMD: The Essential Elements Because angle steer is not always available, there have
Appropriate Ultrasound Technology been a significant number of studies that have settled for an
FMD assessed by Doppler ultrasound has emerged as the insonation of 60.24 26 However, it should be noted that a
most popular clinical research method of assessing vascular 60 angle of insonation is, in fact, the best of the worst
endothelial function, likely because of the relatively simple angles that should be acceptable, and although this value
methodology and noninvasive nature (Figure 1). However, itself introduces some error, this error is still far less than
the appropriate ultrasound equipment, in conjunction with the higher degrees of insonation.2729 The significant impact of
high level of skill required, is essential for accurate and the angle of insonation determination of blood velocity and
reliable measurements, as detailed below. the subsequent calculation of blood flow may be seen in the
Doppler shift equation:
High-Resolution, Multifrequency Linear Ultrasound
v
Doppler Probe (1) D2o cos
The accurate measurement of FMD is highly dependent on c
identification of a defined arterial wall, which requires a where D is the Doppler shift of the reflected ultrasound, o
highly resolved ultrasound image. Each ultrasound probe is is the transmitted frequency, v is the blood velocity, c is the
classified according to a frequency range in megahertz, which sound velocity in tissue, and is the insonation angle
is inversely proportional to the depth of optimal imaging. between the ultrasound beam and the velocity vector. To
Figure 2 demonstrates the quality of B-mode images using illustrate the impact of insonation angle on the measurement
different frequency probes in the brachial artery at a depth of of blood velocity, Table 1 displays the velocity, blood flow,
2 cm. It is clear that. for superficial vessels, a 10- to and subsequent error among different angles of insonation
14-MHz linear array probe, currently considered high reso- studied sequentially in 5 individuals. The velocities associ-
lution, is optimum. However, as recommended previously,4 ated with 70 and 80 angles are significantly (P0.05)
it is also apparent that a much lower frequency probe will elevated when compared with the standard 60 insonation
allow the detection of changes in diameter in all but the most angle; however, only the blood flow calculated from an
superficial of vessels. insonation angle of 80 is significantly augmented when
 Harris et al FMD Tutorial 1077

Ultrasound Technology
Intensity Weighted Insonation Angle
Duplex Mode ECG Gaiting High Resolution Probe
Spectra Correction

Subject Preparation
Tobacco Menstrual Prior Fasted Acclimation Repeated
Vitamins Medications Caffeine
Use Phase Exercise State Phase Measurements

Baseline Measurements
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Arterial Diameter Blood Velocity

Vascular Occlusion
Cuff Position Cuff Duration

Reactive Hyperemia
Temporal Kinetics of Arterial Diameter and Blood Velocity Calculation of Shear Rate

FMD Analysis
Edge Detection Software FMD Calculation FMD Normalization

Figure 1. Schematic of the essential elements for the ultrasound assessment of FMD.

compared with 60. Although an insonation angle of 60 is Intensity-Weighted Velocity Calculations

recommended, the greatest angle typically accepted in liter- The simplest method of assessing blood velocity uses the
ature is 70.26 outer envelope of the Doppler spectra to determine mean
peak velocity, whereas the slightly more complex approach
ECG Gating integrates the area under this envelope to calculate mean peak
Depending on pulse pressure and vascular stiffness, arterial velocity. However, neither of these calculations accurately
diameter may vary quite considerably across a single cardiac reflects the complex and varying range of velocities and their
cycle.30 In some subjects, the change in diameter may be as relative distributions within the Doppler spectra. Therefore,
much as 1 mm, which, if unaccounted for, may completely to accurately assess blood velocity it is recommended that
confound the assessment of FMD. Most ultrasound Doppler intensity-weighted calculations of the time-averaged mean be
systems have an integrated ECG facilitating the assessment of performed to most accurately reflect the contribution from
diameter according to the cardiac cycle (eg, end diastole). red cells moving at differing speeds within the vessel. Basing
However, if this feature is not available on the ultrasound velocity measurements on the peak envelope of the Doppler
system itself, an external ECG can be used to trigger an spectra will not only overestimate the actual blood velocity
external image capture/analysis system.31 but will also yield subsequent calculations of shear profiles
 1078 Hypertension May 2010

Therefore, subjects should abstain from vitamin supplemen-

tation for 72 hours before FMD assessment. Although more
difficult to control, it should be noted that a diet high in
naturally occurring antioxidants may also influence the re-
sults of an FMD study.36

Medications
Because many medications have both direct and indirect
vascular effects, if possible, subjects should refrain from
taking all medications for 4 half-lives of the drug before
FMD measurements.4 In particular, special attention should
be paid to medications that target the cardiovascular system
(ie, -blockers, nitrates, and calcium channel blockers), and
although cessation of these medications may not be feasible,
their potential to confound the results should at the very least
be recognized and documented. On the basis of the half-life
of nonsteroidal anti-inflammatory agents and aspirin, it is
Figure 2. The image quality of B-mode images using different recommended that these be discontinued 1 and 3 days,
frequency linear probes. A, 6 MHz; B, 9 MHz; C, 10 MHz; and respectively, before an FMD measurement.
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D, 12 MHz. The magnification illustrates both the intima to

intima (I-I) and the media to media (M-M) interfaces. Note that Tobacco Use
although there is definitely a visible layer among all of the Smoking is a classic modifiable risk factor of cardiovascular
images above, using at least a 10-MHz probe offers a very clear
disease that has been documented to attenuate endothelial
identification of the endothelium.
function.37 In addition, even the exposure to second-hand
smoke has been shown to attenuate FMD.38 Thus, it is
and blood flows that are inaccurate. If consistently used recommended that subjects refrain from both smoking and
throughout the investigation, the peak velocity envelope smoke exposure for 12 hours before FMD measurements.
approaches do offer a surrogate for true mean velocity
measures, although results may conflict with others in the Caffeine
literature using intensity-weighted velocity calculations.32 In Although there are other pharmacologically active beverages,
summary, an ultrasound system offering Duplex mode, angle coffee is the most common source of caffeine. Not only does
steer and insonation angle correction, intensity-weighted caffeine inhibit soluble guanylate cyclase, a step in the
spectra measurements, ECG monitoring, and a high- NO-mediated process that results in vasodilation,39 caffein-
resolution linear array probe is optimal. ated coffee has been documented to attenuate FMD.40 Ac-
cordingly, caffeine ingestion should be avoided for 12
Subject Preparation hours before FMD testing.
To ensure an accurate measurement of FMD, there are several
subject-specific factors worth consideration. Menstrual Phase
The increased endogenous production of estrogen, concur-
Vitamin Supplementation rently with progesterone, across the menstrual cycle has been
Just as the in vivo pro-oxidant and antioxidant balance plays documented to increase endothelial NO synthase activity41
a clear role in vascular endothelial function,33,34 there is direct and antioxidant capacity42 in both human and animal models,
evidence of a reduction in circulating free radicals after oral thus potentially influencing the vasodilatory response. Con-
antioxidant supplementation (vitamin C, vitamin E, and sequently, when studying premenopausal women, measure-
-lipoic acid).33 In addition, the intra-arterial administration ments should be performed at the same time of the menstrual
of ascorbic acid has been documented to augment FMD.35 cycle. To minimize the impact to these hormonal changes or

Table 1. Evidence of Alterations In Blood Velocity and Blood Flow as a Consequence of Insonation Angle
Theoretical

Insonation Angle Velocity, cm/s Blood Flow, mL/min % From 60 FMD, % Shear, s1 FMD/Shear
40 3.520.46 24.88.3 49 7.0 33.52 0.21
50 4.240.57 29.59.9 25 7.0 40.38 0.17
60 5.270.50 36.711.1 0 7.0 50.19 0.14
70 7.971.07* 55.518.7 51 7.0 75.90 0.09
80 15.692.12* 109.436.8* 197 7.0 149.43 0.05
Values are meanSD unless otherwise specified. This table documents the actual blood velocity and blood flow as functions of
different insonation angles and the theoretical degree of potential error associated with different angles of insonation that will occur
when normalizing FMD for shear.
*Data are significant (P0.05) from 60.
 Harris et al FMD Tutorial 1079

when the research focus is on sex differences, menses (days Baseline Measurements
1 to 7 of the menstrual cycle) offers the lowest attainable Once the investigator confirms that the subject has estab-
levels of both estrogen and progesterone in women and is, lished a resting state (ie, several repeated and consistent
therefore, the optimum time for FMD studies.4,43 measurements of blood pressure, blood velocity, and arterial
diameter), baseline measurements should be performed. In
Previous Exercise/Rested State addition, blood velocities provide an indication that a true
A single bout of exercise has been documented to improve resting state has been achieved and act as the starting point for
FMD in apparently healthy adults,44 overweight men,25 and the shear rate AUC calculations. The collection of accurate
postmenopausal women.45 Therefore, it is important to be baseline diameters is essential for the valid FMD and shear
cognizant of the subjects physiological state; thus, it is rate calculations.
recommended that subjects abstain from exercise for 12
hours before an FMD measurement. Baseline Arterial Diameter
The determination of baseline arterial diameters plays a
Fasted State pivotal role in the calculation and assessment of FMD. There
There is considerable evidence describing the impact of the is evidence to support similar diameters between resting and
postprandial state on the FMD response. Indeed, the con- cuff occlusion conditions; however, recent data have emerged
sumption of a single high-fat and high-carbohydrate meal has to indicate the impact of age50 and cuff duration51 on cuff
been shown to attenuate FMD in apparently healthy sub- occlusioninduced changes in arterial diameter. In addition,
jects46,47 and in patients with type 2 diabetes mellitus,48 in there is evidence to indicate a systemic difference in vascular
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which oxidative stress and hyperglycemia, respectively, have endothelial function that appears to depend on the initial size
been implicated. In contrast, it has been documented that the of the artery.52 In an effort to standardize the FMD method-
ingestion of a low-fat meal (ie, a corn flake cereal with ology, it is recommended that 10 cardiac cycles be used in
skimmed milk) does not influence the FMD measure- the calculation of baseline diameter. Because FMD is based
ment.46,47 Therefore, it is recommended that FMD assess- on change in diameter, within reason, the actual borders (eg,
ments are performed under fasting conditions; however, if intima-media or media-adventitia; Figure 2) that are used to
fasting is not possible, a standardized low-fat meal may be determine the baseline and subsequent diameter are not as
important as the need for consistency from baseline until
consumed before the FMD measurement.
maximal dilation. This having been said, it should be recog-
Adequate Acclimatization nized that measuring from adventitia to adventitia rather than
Because the goal of the FMD measurement is to compare the intima to intima will yield larger values for diameter (not
peak vasodilatory response with the baseline diameter, it is actually representative of the vessel lumen), reducing the
important that a true baseline be accurately assessed. There- percentage of FMD change (because the baseline will be
fore, before an FMD test, it is recommended that subjects larger) and shear rate documented for the vessel being
remain in the position in which the study will be performed studied.
(ie, supine, semisupine, or seated) for 20 minutes in a quiet,
Baseline Blood Velocity
climate controlled room (22C to 24C) to control for orthostatic Blood velocity at rest plays an important role in the calcula-
changes. In addition, a separate familiarization visit of the tion of the shear response to cuff release, especially if using
procedures is recommended to limit stress-induced sympathetic the AUC approach to assess shear rate.53 With the growing
activity on the day of actual measurement. recognition that shear stress is the predominant stimulus for
the FMD response, accurate assessment of resting blood
Repeated Measurements
With respect to either having to repeat an FMD test or the velocity is essential. Even at rest, in an unperturbed scenario,
blood velocity over time can vary substantially (often because
nature of the study design (ie, repeated measures), it has been
of heart rate variability); therefore, it is recommended that
documented that multiple FMD tests can be validly per-
baseline blood velocity be averaged over at least a 10- to
formed if 30 minutes separates each measurement.24 How-
20-second period.54 In subjects who reveal a clear respiratory
ever, an important phenomenon to acknowledge is that FMD
arrhythmia, this may need to be extended significantly,
measurements exhibit diurnal variation,49 and, thus, compar-
depending on respiratory rate, to reflect a true average basal
isons between and within subjects should be performed as blood velocity.
consistently as possible with regard to the time of day. In addition to the clear need to optimize the insonation
In summary, appropriate subject preparation is essential to angle to accurately assess baseline blood velocities, place-
the successful ultrasonic assessment of FMD. Therefore, ment and size of the sample volume (gate width) are of
strict compliance in terms of limiting vitamin supplementa- critical importance, especially when velocities are low. Fig-
tion, cessation (or at least documentation) of medications, ure 3 illustrates the impact of the Doppler sample gate size on
tobacco and caffeine use, phase of the menstrual cycle, the measurement of resting blood velocity and blood flow. In
previous exercise, and being fasted and rested before the accordance with the hydraulic properties of Newtonian fluids,
FMD measurement is essential. Finally, if repeated measure- Figure 3 identifies 80% overestimation of volume flow
ments are necessary, an adequate amount of vessel recovery when smaller sample volume sizes are used along the center
time should be allotted while also recognizing that there is of the vessel (Figure 3A, top) compared with a sample
diurnal variation in the FMD response. volume size spanning intima-to-intima (Figure 3C, bottom),
 1080 Hypertension May 2010

very least between before and after cuff release within

individual subjects and repeated measurements on the same
subject.
In summary, the baseline measurements are extremely
important components of an FMD study, because these
assessments are built on after cuff release. Therefore, the
accurate assessment of an average diameter and concurrent
blood velocities, with appropriate sample volume, for 10
cardiac cycles is recommended before vascular occlusion
(cuff inflation).

Vascular Occlusion
The initial stimulus for any FMD test relies on temporary
vascular occlusion, which creates a region of ischemic tissue
distal to the point of occlusion.1 The metabolic byproducts of
cellular respiration, in the absence of circulating blood,
promote an increase in vascular conductance that allows a
robust hyperemia on eradication of the upstream occlusion.
This reactive hyperemia, and the associated shear stress
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experienced by the conduit vessels upstream from the area of

occlusion, is the primary stimulus for FMD. Accordingly,
both cuff position (proximal cuffgreater volume of tissue
experiencing ischemiagreater hyperemia) and the duration
of occlusion (longer occlusiongreater degree of ische-
miagreater hyperemia) play integral roles in shear-mediated
vasodilation.

The Cuff
The size of the cuff used for vascular occlusion should be
appropriate for the area being occluded. Although a conven-
tional hand-inflated cuff is adequate, it is both convenient and
methodologically sound to almost instantaneously inflate and
deflate the cuff, which can be achieved using a commercially
available rapid (0.3-second) cuff inflator. It should be noted
that a potential down fall of this rapid inflation approach is
the jolt that can accompany both inflation and deflation,
which can be avoided by supporting the arm in such a way
that there is adequate space under the arm for the cuff to
inflate and deflate.

Cuff Position
Although there is no consensus regarding the placement of
the occlusion cuff relative to the site of measurement, there is
growing support in favor of placement distal to the ultrasound
probe, because this approach is thought to yield a predomi-
nantly endothelium-dependent vasodilation.55 Positioning the
cuff proximal to the imaging site elicits a greater peak
hyperemic response and subsequent FMD56 likely attributed
to ischemia-induced hypoxia in the area being imaged. In
Figure 3. The determination of blood velocity and blood flow addition, the decreased hyperemic decay observed after cuff
using different placements of the Doppler sample gate. A, Outer;
B, middle; C, inner. Note the difference in velocity and blood deflation proximal to the site of measurement (Doppler
flow among the different placements of the sample gate. probe) suggests that mechanisms in addition to NO-mediated
vasodilation play a role in this scenario.51,55
because of the laminar nature of blood flow and the reduction Cuff Duration
in blood velocity near the vessel wall. Thus, it is recom- Although it has been documented previously that a 10-minute
mended that the sample volume be as wide as possible but cuff occlusion results in no greater maximal arterial dilation
without encompassing the vessel walls and allowing for a than a 5-minute occlusion,4 for the sake of consistency and
slight margin for error in case of movement (Figure 3B). subject comfort it is recommended that a 5-minute suprasys-
Although the width of the gate will vary between laboratories, tolic cuffing period (200 to 250 mm Hg) be used for FMD
emphasis should be placed on maintaining consistency at the tests. Although the link among vascular function in the
 Harris et al FMD Tutorial 1081

brachial artery, NO, and the duration of cuff occlusion is still is influenced by the width of the sample volume and the
a matter of some debate,13,21 recent evidence supports the use placement of this gate within the vessel. This is the conse-
of a 5-minute over a 10-minute occlusion, because sustained quence of the parabolic velocity profile within unbranched
(5-minute) occlusion may include more non-NO, ischemia- sections of conduit vessels. This same concept has an impact
induced vasodilators.57 Consistent with this, our laboratory on the calculation of shear rate, which is derived from the
has recently documented a 50% greater brachial artery Poiseuilles law, dependent on Doppler sample volume size
vasodilation after 10 versus 5 minutes of occlusion, even after and placement: (1) large, centered sample volume: 8mean
normalizing FMD for shear rate,31 suggestive of nonendothe- blood velocity/internal diameter; or (2) small, centered sam-
lium-dependent, NO-mediated vasodilators playing a signif- ple volume: 4mean blood velocity/internal diameter.
icant role after longer bouts of ischemia. The difference in numerator (8 versus 4) of this calculation
In summary, when performing an FMD test, the cuff being explained by the failure to account for slower-moving
should be the appropriate size for the limb being studied, red cells at the edge of vessel and, therefore, a bias toward an
positioned distal to the ultrasound probe, and inflated 25 to artificially elevated mean blood velocity as the sample
50 mm Hg above systolic arterial pressure for 5 minutes to volume becomes smaller but still located in the center of the
elicit a reactive hyperemic stimulus that is considered to be vessel. On the basis of this information, it is recommended
predominantly endothelium mediated and NO dependent. that the Doppler sample volume be kept wide and when shear
rate is calculated the factor of 8 be used in the numerator of
Reactive Hyperemia (Postcuff Release) Measurements
The measurements after vascular occlusion (ie, postcuff this equation.
In summary, it is recommended that both diameter and
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release) are just as important, if not more so, than the baseline
measurements. The following observations and recommenda- velocity data be acquired for 10 seconds before cuff release
tions apply to measurements during the postcuff release time and continue these data collection for 2 minutes postre-
period. lease. This method will not only allow for the documentation
of the true peak diameter but it will also allow the quantitative
Temporal Kinetics of Arterial Diameters and analysis of shear AUC, the stimulus thought to be predomi-
Blood Velocities nantly responsible for the FMD response. In addition, docu-
As discussed above, it is recommended that Duplex mode on menting the time to peak vasodilation may more appropri-
the ultrasound system be used. To ensure that no anomalies in ately assess endothelial function when making comparisons
arterial diameter occur during cuff occlusion and to capture among different groups and/or clinical populations.
the immediate hyperemic response, it is recommended that
postcuff measurements be initiated 10 seconds before cuff FMD Analyses
release. Although the peak velocity occurs within the first 15 Recently, it has become apparent that the measurement of
seconds, the peak vasodilation can be expected to occur 45 to FMD may not be as simple as assessing vessel diameter both
80 seconds after cuff release and may differ between popu- before and after cuff release and reporting a percentage of
lations.58 Thus, it is also now recommended that the true peak increase in vessel caliber. Indeed, over the past 2 decades,
diameter be determined on an individual basis (not simply the both the methodology and analysis of FMD have received
diameter in a given window of time) and the time to peak significant attention, evolving into what are recommended
vasodilation be reported. today. For example, it is now acknowledged that, when using
To capture the kinetics of reactive hyperemia-induced the traditional percentage of change calculation, the initial
shear and subsequent vasodilation, it is recommended that baseline diameter has the potential to introduce mathematical
blood velocity and diameter measurements be performed for bias into the FMD assessment, with smaller vessels appearing
2 minutes after cuff release. Because the velocity profile more reactive and vice versa.7 Therefore, it is now recom-
after occlusion is characterized by a parabolic shape with mended that, in addition to FMD expressed as a percentage,
exponential decay, it is generally agreed on that the integral researchers document baseline diameters, absolute change in
of shear rate over time (ie, AUC) is the optimal method to diameter, and shear rate (AUC).
quantify the accumulated shear that contributes to the FMD
response.53 AUC is conventionally calculated using the trap- Edge Detection Software
ezoidal rule, according to the following equation: The use of edge detection software for offline analysis is
recommended for the measurement of baseline and postcuff
(2) {yi[ x(i1)xi](1/ 2)[ y(i1)yi][ x(i1)xi]} release diameters. Using this approach facilitates more objec-
tive and accurate diameter measurements and also permits
where x is time, y is shear, xi is initial time point, and yi is
synchronization with the ultrasound system and ECG to allow
initial blood velocity.
sequential end-diastolic images to be stored, avoiding arti-
Although Duplex mode is required to calculate the total
facts attributed to pulse-related changes in vessel diameter.
shear rate (AUC), the previous recommendation to capture
Currently, the most commonly used, commercially available
the peak hyperemic velocity during the first 15 seconds4
edge detection software is that developed by Medical Imag-
before switching back to 2D imaging is still a feasible
ing Applications LLC. This edge detection software has been
method, acknowledging the limitations of such an approach.
independently validated59 and is now commonly found in the
Calculation of Shear Rate literature.25,31,35,60 62
As described in the baseline velocities section and illustrated If edge detection software is unavailable, it is recom-
in Figure 3, the measurement of blood velocity with Doppler mended that data (diameter and velocity) be collected every 4
 1082 Hypertension May 2010

Table 2. Absolute Difference and Variability Between Manual and Software Evaluations of FMD Determinants
Baseline Diameter, cm Peak Diameter, cm FMD, %

Subject Manual Software Difference, cm CV, % Manual Software Difference, cm CV, % Manual Software Difference, cm CV, %
1 0.42 0.4000 0.015 2.6 0.44 0.4200 0.020 3.3 6.02 5.00 1.02 13.1
2 0.43 0.4497 0.022 3.6 0.45 0.4689 0.019 2.9 5.26 4.27 0.99 14.7
3 0.40 0.3951 0.002 0.4 0.41 0.4027 0.007 1.3 3.14 1.92 1.22 34.1
4 0.34 0.3475 0.007 1.5 0.35 0.3595 0.010 1.9 2.94 3.45 0.51 11.3
5 0.31 0.3200 0.010 2.2 0.35 0.3511 0.001 0.2 12.90 11.42 1.48 8.6
6 0.30 0.3029 0.003 0.7 0.32 0.3244 0.004 1.0 6.67 8.13 1.47 14.0
7 0.33 0.3200 0.010 2.2 0.37 0.3600 0.010 1.9 12.12 12.50 0.38 2.2
8 0.24 0.2400 0.000 0.0 0.27 0.2900 0.020 5.1 12.50 20.83 8.33 35.4
9 0.29 0.2833 0.009 2.3 0.31 0.3100 0.000 0.0 5.98 9.41 3.43 31.5
10 0.33 0.3204 0.006 1.4 0.36 0.3483 0.012 2.3 10.20 8.71 1.50 11.2
11 0.29 0.2733 0.017 4.2 0.31 0.2900 0.020 4.7 6.90 7.41 0.51 5.1
12 0.25 0.2500 0.003 0.9 0.27 0.2600 0.010 2.7 6.58 5.41 1.17 13.8
13 0.38 0.3900 0.010 1.8 0.39 0.3974 0.007 1.3 2.63 2.03 0.60 18.3
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14 0.38 0.3800 0.000 0.0 0.38 0.3807 0.001 0.1 0.00 0.00 0.00 0.0
15 0.30 0.3000 0.000 0.0 0.31 0.3100 0.000 0.0 3.33 3.33 0.00 0.0
Mean 0.33 0.3315 0.001 1.6 0.35 0.3515 0.001 1.9 6.48 6.92 0.4 15.2
Note that there is a greater accuracy of diameter measurements and the subsequent determination of FMD using software analysis.

seconds for the first 20 seconds after cuff release, followed by is too short (ie, 3 seconds) inevitably increases the noise of
every 10 seconds for the remaining 2-minute data collection the measurement and may result in identification of an
period. Table 2 illustrates the variability and absolute differ- aberrant value for peak diameter. Accordingly, it is recom-
ences in baseline diameter, peak diameter, and calculated mended that the peak diameter be determined over as short a
FMD between careful manual (calipers on the ultrasound) period of time as possible (ie, 5 seconds) but never relying on
and edge detection software evaluation in a range of subjects. peak diameter data that are obtained from the average of 3
Although strong correlations and low variability exist for measurements (ie, 3 cardiac cycles). The data-smoothing time
baseline diameter (r0.98; coefficient of variation [CV]: period should be reported and blood velocities should be
1.9%), peak diameter (r0.98; CV: 1.9%), and calculated analyzed during the same time frame as the diameters (ie,
FMD (r0.89; CV: 15.2%), it does appear that using edge Duplex mode).
detection software not only provides a more robust and
sensitive assessment of FMD, it also removes any subjective Normalization of FMD (FMD/Shear)
error component from the data analysis. Because FMD is thought to be evoked by shear stress and,
thus, proportional to reactive hyperemia, consideration of the
Calculation of FMD heterogeneity of blood flow responses across subjects de-
The calculation of FMD as a percentage change uses the peak serves greater attention than it received at the outset of such
diameter in response to reactive hyperemia in relation to the endothelial function measurements.4 Indeed, it has been
baseline diameter and is calculated using the following suggested recently that FMD should be normalized by divid-
equation: ing the percentage of FMD by shear rate (AUC).53 Although
peak diameterbaseline diameter this mathematical correction for shear stimulus is theoreti-
(3) FMD(%) cally simple, experimental evidence supporting normalization
baseline diameter
is equivocal. In the study of vascular aging, it has been
and when multiplied by 100, FMD is expressed as a percent-
age of change in the vessel caliber. Table 3. Difference in FMD and Determinants Using 3-, 5-,
It is recommended that the average diameter assessed and 10-Second Data Smoothing Averages
during end diastole (identified by the R wave on the ECG) Variable 3 Seconds 5 Seconds 10 Seconds
over 10 cardiac cycles be used to represent the baseline
Baseline diameter 3.250.15 3.250.15 3.250.15
diameter. With the introduction of the edge detection soft-
ware, some debate has developed as to the optimum time Peak diameter 3.450.15 3.430.15* 3.420.15*
resolution necessary to accurately determine peak diameter.58 FMD, % 6.70.9 6.00.8 5.60.7*
The differences in FMD and determinants using 3-, 5-, and Time to peak, seconds 446 434 446
10-second data smoothing averages are presented in Table 3. No. of frames 2.40.3 4.00.4* 7.70.7*
Indeed, if diameters are averaged (data smoothing) over some Data are presented as meanSEM.
period of time (ie, 10 seconds), the exact peak will lose *Data are significant (P0.05) from 3 seconds.
resolution. However, data smoothing with a time period that Data are significant (P0.05) from 5 seconds.
 Harris et al FMD Tutorial 1083

reported that healthy older adults have a preserved endothe-

lial function when FMD is normalized for their reduced
14
A YOUNG:
R = 0.83
postcuff release shear rates.32 In addition, Padilla et al62 12
YOUNG

FMD (% Diameter Change)

demonstrated that normalizing FMD for shear rate (AUC) OLD
10
eliminates the influence of differing shear profiles created by
varying periods of cuff ischemia. Additional data to support 8
normalization have been indicated by the removal of limb-
6
specific differences in FMD when shear rate was used to Peak Shear

Shear Rate
OLD:

Diameter
normalize the FMD response.63 In contrast, a recent study has 4 R = 0.23
concluded that normalizing FMD for shear is age dependent
2
and only appropriate when investigating young adults.64
Therefore, the use of Duplex ultrasound and the subsequent 0
Time

ability to assess FMD, measure reactive hyperemia, and 200 400 600 800 1000 1200 1400 1600 1800
calculate shear AUC are emerging as important components Shear Rate (Peak)

of FMD measurements made with ultrasound Doppler. In

addition, reactive hyperemia alone has been documented to
14
B YOUNG:
R = 0.90
have high clinical prognostic value.65 12

FMD (% Diameter Change)

In statistical terms, the appropriate method by which to
10
take differing shear rates into account during an FMD study
Downloaded from http://hyper.ahajournals.org/ by guest on May 29, 2017

OLD:
is complex. To legitimately apply such a correction factor to 8 R = 0.79
a variable, the relationship between the 2 variables must
6
satisfy 3 assumptions: (1) a significant correlation; (2) the y Peak Diameter

Shear Rate

Diameter
intercept of this relationship must be 0; and (3) the data 4
should be normally distributed. From our own experience and
that of others (Figure 4 and References 53 and 64), the 2

relationship between vessel dilation and shear rate often 0

Time

violates 1, if not all 3, of these assumptions, leaving serious 0 10000 20000 30000 40000 50000 60000
doubt as to whether a simple mathematical normalization Shear Rate AUC (to peak diameter)

should be categorically performed. If, as has been suggested,

there is simply a modest correlation between FMD and shear
14
C YOUNG:
R = 0.83
and the shear stimulus differs between independent variables, 12
FMD (% Diameter Change)

the proper method of taking into account the covariance of 10

shear rate with FMD may be through the ANCOVA,66
OLD:
although, as of yet, this method is also not completely 8 R = 0.68
accepted.
6
Figure 4 illustrates the relationships between FMD and
Shear Rate

Diameter
peak shear, shear AUC up to the time of peak dilation, and 4
total shear AUC (2 minutes) in both young and old subjects.
Although all of the relationships for the young population 2

appear to be very strong, it is important to note that only the 0

Time

shear AUC until peak vasodilation (Figure 4, plot B) yields 0 10000 20000 30000 40000 50000 60000
the strongest relationship in both age groups. These data are Shear Rate AUC (Total; 2 min)

in agreement with previous work53 and provide further Figure 4. The relationships between FMD and different assess-
evidence that shear rate (AUC) until the time of peak dilation ments of shear rate to be considered when normalizing FMD. A,
FMD vs peak shear; B, FMD vs shear AUC until peak diameter;
may be the most appropriate method of quantifying shear C, FMD vs total shear AUC for the entire 2 minutes. Inlays for
forces. each panel illustrate the corresponding shear rate (shaded) used
In summary, edge detection software has been indepen- in the analysis.
dently validated and is recommended for the measurement of
arterial diameter. To standardize the FMD technique, the lation) and FMD data also be readily available to allow for
recommended procedure for obtaining diameters is through alternative analyses or interpretations. In addition, the time
continuous digital data recording and offline analysis using it takes to obtain peak vasodilation may be an important
edge detection software. To identify and calculate the FMD indicator of stimulus sensitivity that should be incorpo-
response, the true peak diameter is obtained and expressed as rated into an evaluation of endothelial function by an FMD
an increase in vasodilation above baseline values. Although test.
normalization of FMD for shear has been embraced by many
researchers, uncertainty currently exists as to how to Conclusion
properly normalize FMD. It is currently recommended that The measurement of FMD is often mistaken as a simple
FMD normalized for shear rate (AUC) be calculated and noninvasive method of assessing vascular endothelial func-
reported but that the raw shear (AUC up to peak vasodi- tion that anyone with access to an ultrasound Doppler can
 1084 Hypertension May 2010

perform.13 However, the appropriate ultrasound technology, 16. Niebauer J, Cooke JP. Cardiovascular effects of exercise: role of endo-
subject preparation, and knowledge of the method are re- thelial shear stress. J Am Coll Cardiol. 1996;28:16521660.
17. Sessa WC. eNOS at a glance. J Cell Sci. 2004;117:24272429.
quired to perform an accurate assessment of endothelial 18. Shimokawa H, Yasutake H, Fujii K, Owada MK, Nakaike R, Fukumoto
function using the FMD technique. The recommendations Y, Takayanagi T, Nagao T, Egashira K, Fujishima M, Takeshita A. The
proposed in this comprehensive tutorial represent the most importance of the hyperpolarizing mechanism increases as the vessel size
decreases in endothelium-dependent relaxations in rat mesenteric circu-
recent advancements in the ultrasonic measurement of FMD
lation. J Cardiovasc Pharmacol. 1996;28:703711.
and are presented in an attempt to standardize this measure- 19. Doshi SN, Naka KK, Payne N, Jones CJ, Ashton M, Lewis MJ, Good-
ment across research sites and to subsequently facilitate the fellow J. Flow-mediated dilatation following wrist and upper arm
use of FMD as a clinically relevant research tool. occlusion in humans: the contribution of nitric oxide. Clin Sci (Lond).
2001;101:629 635.
20. Mullen MJ, Kharbanda RK, Cross J, Donald AE, Taylor M, Vallance P,
Sources of Funding Deanfield JE, MacAllister RJ. Heterogenous nature of flow-mediated
We were supported by National Institutes of Health grant PO1-HL- dilatation in human conduit arteries in vivo: relevance to endothelial
0918830, Tobacco-Related Disease Research Program grant 15RT- dysfunction in hypercholesterolemia. Circ Res. 2001;88:145151.
0100, the Parker B. Francis Family Foundation, the American Heart 21. Tschakovsky ME, Pyke KE. Counterpoint: flow-mediated dilation does
Association Scientist Development Grant 08535209N, and the Salt not reflect nitric oxide-mediated endothelial function. J Appl Physiol.
Lake City Veterans Affairs Medical Center Geriatric Research, 2005;99:12351237.
Education, and Clinical Center. 22. Pyke K, Green DJ, Weisbrod C, Best M, Dembo L, ODriscoll G,
Tschakovsky M. Nitric oxide is not obligatory for radial artery flow-
mediated dilation following release of 5 or 10 min distal occlusion. Am J
Disclosures Physiol. 2010;298:H119 H126.
None.
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23. Mitchell GF, Parise H, Vita JA, Larson MG, Warner E, Keaney JF Jr,
Keyes MJ, Levy D, Vasan RS, Benjamin EJ. Local shear stress and
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 Ultrasound Assessment of Flow-Mediated Dilation
Ryan A. Harris, Steven K. Nishiyama, D. Walter Wray and Russell S. Richardson

Hypertension. 2010;55:1075-1085; originally published online March 29, 2010;

doi: 10.1161/HYPERTENSIONAHA.110.150821
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