Open Journal of Clinical Diagnostics, 2011, 1, 15-21
doi:10.4236/ojcd.2011.13004 Published Online December 2011 (http://www.SciRP.org/journal/ojcd/
OJCD
).
Published Online December 2011 in SciRes. http://www.scirp.org/journal/OJCD
Optimization of ultrasound assessments of arterial function
Lee Stoner1,2*, Cary West 2, Danielle Morozewicz Cates2, Joanna M. Young3
1School of Sport and Exercise, Massey University, Wellington, New Zealand;
2Department of Kinesiology, University of Georgia, Ramsey Center, Athens, USA;
3Lipid and Diabetes Research Group, Diabetes Research Institute, Christchurch, New Zealand.
Received 16 September 2011; revised 18 November 2011; accepted 18 November 2011.
ABSTRACT
Ultrasound technology is widely used to make as-
sessments of arterial function. The delicate nature of
these measurements requires that sources of errors
are limited. Therefore, the aim of this study was to
assess variability due to probe selection and optimi-
zation settings. Methods: Ten healthy 20 - 26 year old
male and female subjects were tested. Brachial artery
size (diameter) was measured thirty times a second
using a B-mode Ultrasound unit equipped with a
high-resolution video capture device. Distension was
calculated using systolic and diastolic diameters. To
assess intersession variability, we made recordings
over twelve minutes; with the probe being removed
and re-positioned every four minutes. To assess vari-
ability due to probe selection and optimization, we
manipulated four parameters: 1) Probe selection (7 -
13 MHz, 5 - 10 MHz, 6 - 9 MHz); 2) Probe frequency
(11 MHZ, 9.6 MHZ, 8 MHz); 3) Measurement loca-
tion (near, center or middle field); And 4) Image
mode (B-mode, duplex-mode). To assess inter-session
variability, three sets of recordings were made for
each probe selection and optimization setting. Results:
Mean diameter ICC’s for inter-session variability,
probe frequency, measurement location, image dis-
play size, and probe selection were 0.99, 0.98, 0.97,
0.99, and 0.90 respectively. Distension ICC’s for
intersession variability, probe frequency, measure-
ment location, image display size, and probe selection
were 0.66, 0.26, 0.62, 0.60, and 0.51 respectively.
Conclusions: Altering probe selection increases
measurement variability to the greatest extent. How-
ever, as long as probe selection and optimization set-
tings are kept constant, our inter-session variability
shows that reliable measurements can be made.
Keywords: Ultrasound; Reproducibility; Diameters; Dis-
tension; Arterial Stiffness
1. INTRODUCTION
Ultrasound is widely used for the diagnostic assessment
of the carotid and peripheral arteries. The elastic proper-
ties of carotid and peripheral arteries are assessed by
studying dynamic properties of the arterial walls. Through
measurement of arterial distention, together with local
blood pressures, indices of arterial stiffness can be cal-
culated. Assessments of arterial stiffness have shown to
predict future cardiovascular complications [1-4].
In order to calculate arterial stiffness, the diameter of
a given artery must be continuously measured across the
cardiac cycle. For a carotid artery, this may entail meas-
urements that range from 8.0 mm to ~ 8.3 mm, a disten-
tion of 0.3 mm. For peripheral arteries, the distention
range will be much lower. Therefore, even small varia-
tions in systolic or diastolic diameters can notably im-
pact distention measurements. For this reason, it is im-
portant to limit possible sources of error. However, the
requirement for standardization of ultrasound technical
settings has not been reported in the literature.
The aim of this study was to assess variability due to
probe selection and optimization settings. To assess
variability due to probe selection and optimization four
parameters were manipulated: 1) Probe selection; 2)
Probe frequency; 3) Measurement location; And, 4) Im-
age mode. To assess inter-session variability, three re-
cordings were made for each parameter.
2. METHODS
2.1. Subjects
Ten healthy 20 - 26 year old male and female subjects
were tested. Informed consent was obtained from the
subjects after they were given a detailed description of
the procedures. The study was approved by the Univer-
sity of Georgia Institutional Review Board. Subjects
were excluded from the study if they demonstrated any
cardiovascular disease health risks or were taking medi-
cations with known vasoactive properties. Subjects were

L. Stoner et al. / Open Journal of Clinical Diagnostics 1 (2011) 15-21 16
asked to abstain from caffeine, high-fat foods, and alco-
hol for 24 hours prior to testing.
2.2. Protocol
Testing commenced following at least 20 minutes of
quiet supine rest. All measurements for a given subject
were made in one sitting. Brachial artery size (diameter)
was measured using a B-mode Ultrasound unit equipped
with a high-resolution video capture device. Diastolic,
systolic and mean diameters were recorded. Recordings
were made using eleven probe selection and optimiza-
tion settings (see Table 1). To assess inter-session vari-
ability, three sets of recordings were made for each
probe selection and optimization setting, with the probe
being removed and re-positioned every four minutes.
The three probes were linear array transducers. To com-
pare probes the highest imaging frequencies were set for
each probe (LA39, 11 MHz; 739, 9 MHz; 546, 6.6 MHz).
Aside from the probe comparison measurements, the
highest resolution probe (LA39) was used. Aside from
the location measurements, images were focused on the
center of the image display field. Aside from imaging
mode measurements, B-mode was used. Care was taken
to ensure that the same portion of the brachial artery was
imaged for all measurements. Subjects were asked to
hold their breath for ten seconds for each recording.
2.3. Diameter Measurements
High-resolution Brightness-mode (B-mode) ultrasound
measurements were made using a GE 400CL duplex
color Doppler unit (GE Medical, Milwaukee, Wisconsin).
The brachial artery of the left arm was measured in the
distal third of upper arm. Care was taken to ensure that
the vessel clearly extended across the entire [un-zoomed]
imaging plane to minimize the likelihood of skewing the
vessel walls. Magnification and focal zone settings were
then adjusted to optimize imaging of the proximal and
distal vessel walls. The image was comprised of 400 ×
400 pixels over an area of 16 × 16 mm, with a pixel
resolution of 0.04 × 0.04 mm. A specialized probe hold-
ing device enabled precise positioning and ensured that
pressure on the artery was minimized. The precise posi-
tion of the ultrasound probe was recorded and marked.
2.4. Diameter Analysis
Moving Picture Experts Group-2 (MPEG-2) recordings
were captured using a Dell Laptop PC equipped with a
video capture device (ADS technologies, Cerritos, Cali-
fornia). Video files collected at 30 frames/second were
converted to Joint Photographic Experts Group (JEPG)
images and subsequently used to make 30 diameter
measurements/second. JPEG images provide comparable
accuracy for ultrasound image measurements compared
Table 1. Probe and optimization setting parameters.
Optimization Setting Sub-Setting
LA39 (7 - 13 MHz)
739 (5 - 10 MHz)
Linear Array Probe
549 (6 - 9 MHz)
11 MHz
9.6 MHz
Probe Frequency
8 MHz
Near Field
Center field
Field Location
Far Field
B-Mode
Imaging Mode
PD-Mode (duplex)
to the Digital Image and Communications in Medicine
(DICOM) standard [5]. Images were measured offline
using semi-automated edge-detection software custom
written to interface with the LabVIEW data acquisition
platform (version 8.1, National Instruments, Austin,
Texas) [6,7]. Custom written Excel Visual Basic code
was used to fit peaks and troughs to diameter waveforms
in order to calculate diastolic, systolic, and mean diame-
ters. The within-session SEM3,1 for diameter measure-
ment with the described set-up is 0.046 mm. The be-
tween-day coefficient of variation is 2.7% for resting
diameter measurements [8].
2.5. Statistical Analysis
Statistical analysis was undertaken using SPSS 13 for
windows (SPSS Inc, Chicago, IL). The single measures
intra-class correlation coefficient (ICC) were calculated
using a two-way mixed effects (absolute agreement)
model where subject effects are random and the optimi-
zation/probe settings fixed. In general, values above 0.75
can be considered to represent excellent reliability, val-
ues between 0.4 and 0.75 represent fair to good reliabil-
ity and values below 0.4 represent poor reliability [9].
Standard error of measurement (SEM) and respective
confidence intervals were calculated using Eq. 1 and Eq.
2:

SEMSD1 ICC (1)

95% CIMeanSEM1.96

(2)
where SD = the sample standard deviation, and ICC = as
calculated above.
Bland-Altman plots were constructed to provide an
indication of systematic bias and random error [10,11].
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L. Stoner et al. / Open Journal of Clinical Diagnostics 1 (2011) 15-21 17
The 95% confidence intervals of limits of agreement
were calculated using Eq. 3:
95%1.96 SDd (3)
where: d = the sample bias (mean difference), and SD =
standard deviation of differences.
3. RESULTS
3.1. W ithin-Ses sion Va riability
Table 2 shows the inter-session variability for diameter
measurements. Single measure ICC values for Dm, Dd,
and Ds show excellent reliability. The %SEM is lowest
for Dd and highest for Ds. The Bland-Altman plot shown
in Figure 3 compares trials 1 and 3. There was no indi-
cation of systemic bias across the three trials. The ICC
for D∆ shows fair to good reliability. The %SEM is no-
tably higher for D∆ than for single diameters. Bland-
Altman plots for D∆ (not shown) do not indicate sys-
temic bias over the three trials.
3.2. Diameter Measurement Variability across
Ultrasound Settings
Table 3 shows the variability for diameter measure-
ments across ultrasound settings. Single measure ICC
values for Dm, Dd, and Ds diameters show excellent reli-
ability. The %SEM is lowest for Ds and highest for Dd.
The ICC for D∆ shows poor reliability. The %SEM is
notably higher for D∆ than for systolic or diastolic di-
ameters.
3.3. Diameter Measurement Variability for Each
Ultrasound Setting
Table 4 shows the variability for diameter measure-
ments for each ultrasound setting. Figures 1 and 2 show
example diameter analysis and resultant waveforms for
LA39 and 546 probes. ICC values for Dm, Dd, and Ds
across probe frequencies show excellent reliability.
Bland-Altman plots (not shown) show a bias for smaller
diameters as the probe frequency decreases.
The ICC for D∆ shows poor reliability. The %SEM is
notably higher for D∆ than for single diameters. The
Bland-Altman plot shown in Figure 4 compares fre-
quencies 11 MHz and 8 MHz for the LA39 probe. There
was no indication of systemic bias across the three probe
frequencies.
ICC values for Dm, Dd, and Ds across measurement
location show excellent reliability. Bland-Altman plots
(not shown) show a bias for smaller diameters as the
measurement location moves from the near- to the
far-side of the ultrasound imaging field. The ICC for D∆
shows fair to good reliability. The %SEM is notably
higher for D∆ than for single diameters. Bland-Altman
plots (not shown) show a bias for larger D∆ as the meas-
Figure 1. Examples of semi-automated diameter analysis on
images collected from (A) LA39 (11 MHz) and (B) 546 (6.6
MHz) probes. Images are captured at a rate of 30 images per
second. The images are identical to those shown in Figure 2.
A. LA39 (11MHz)B. 546 (6.6MHz)
3.7
3.8
3.9
4.0
0123
Time (s ecs)
Diamet er ( m m )
3.7
3.8
3.9
4.0
0123
Time (s ecs)
Diamet er ( m m )
Figure 2. Example of images collected from (A) LA39 (11
MHz) and (B) 546 (6.6 MHz) probes. Images were taken dur-
ing diastole and illustrate the first image used for analysis as
shown by the corresponding graphs above the images.
urement location moves from the near- to the far-side of
ultrasound imaging field.
ICC values for Dm, Dd, and Ds across imaging mode
show excellent reliability. Bland- Altman plots (not
shown) show a bias for smaller diameters when imaging
in PD versus B-mode. The ICC for D∆ shows fair to
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L. Stoner et al. / Open Journal of Clinical Diagnostics 1 (2011) 15-21 18
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good reliability. The %SEM is notably higher for D∆
than for single diameters. Bland- Altman plots (not
shown) show no indication of bias.
ICC values for Dm, Dd, and Ds across probe selection
show excellent reliability. Compared to the other opti-
mization settings probe selection results in the lowest
ICC and highest %SEM. The Bland-Altman plot shown
in Figure 5 compares probes LA39 (11 MHz) and 539
(6.6 MHz). The 539 probe results in smaller diameters
than for LA39 and 739 (9 MHz) probes. The LA39 and
739 probes are more comparable. The ICC for D∆ shows
fair to good reliability. The %SEM is notably higher for
Table 2. Inter-session diameter measurement variability.
Dm Ds Dd D∆
T1 (mm) 4.143 (0.545) 4.189 (0.518) 4.076 (0.487) 0.113 (0.053)
T2 (mm) 4.149 (0.493) 4.196 (0.495) 4.076 (0.492) 0.120 (0.054)
T3 (mm) 4.148 (0.506) 4.198 (0.509) 4.081 (0.486) 0.117 (0.066)
Mean (mm) 4.147 (0.513) 4.194 (0.506) 4.078 (0.488) 0.117 (0.050)
ICC 0.992 0.991 0.996 0.661
SEM3,1 (mm) 0.046 0.048 0.031 0.029
SEM (%) 1.107 1.144 0.757 25.171
LCI (mm) 4.057 4.100 4.017 0.059
UCI (mm) 4.237 4.288 4.138 0.174
Diastolic, systolic, and mean diameters, and distension for each four-minute interval. Values are mean (SD).
Table 3. Diameter measurement variability across ultrasound settings.
Dm Ds Dd D∆
LA39 (mm) 4.121 (0.504) 4.161 (0.505) 4.089 (0.504) 0.072 (0.043)
739 (mm) 4.140 (0.539) 4.176 (0.542) 4.107 (0.530) 0.069 (0.046)
546 (mm) 4.040 (0.605) 4.101 (0.604) 4.002 (0.619) 0.099 (0.044)
11 mhz (mm) 4.183 (0.553) 4.252 (0.499) 4.168 (0.504) 0.084 (0.042)
9.6 mhz (mm) 4.127 (0.516) 4.191 (0.514) 4.090 (0.510) 0.101 (0.064)
8.2 mhz (mm) 4.109 (0.533) 4.148 (0.554) 4.070 (0.537) 0.078 (0.043)
Left (mm) 4.205 (0.487) 4.230 (0.502) 4.175 (0.482) 0.055 (0.050)
Cent. (mm) 4.179 (0.473) 4.209 (0.469) 4.145 (0.471) 0.064 (0.044)
Right (mm) 4.144 (0.516) 4.186 (0.527) 4.109 (0.510) 0.077 (0.047)
B (mm) 4.182 (0.476) 4.226 (0.477) 4.156 (0.481) 0.071 (0.042)
PD (mm) 4.151 (0.501) 4.191 (0.521) 4.127 (0.501) 0.064 (0.067)
Mean (mm) 4.144 (0.046) 4.190 (0.043) 4.113 (0.051) 0.077 (0.014)
ICC 0.915 0.918 0.914 0.372
SEM3,1 (mm) 0.013 0.012 0.015 0.011
SEM (%) 0.320 0.292 0.360 14.26
LCI (mm) 4.118 4.166 4.084 0.055
UCI (mm) 4.170 4.214 4.142 0.098
Diastolic, systolic, and mean diameters, and distension for each ultrasound setting. Values are mean (SD).

L. Stoner et al. / Open Journal of Clinical Diagnostics 1 (2011) 15-21 19
Table 4. Diameter measurement variability for each ultrasound setting.
Mean (mm) ICC SEM3,1 (mm) SEM (%) 95%CI (mm)
Dm 4.100 (0.532) 0.902 0.167 4.065 (3.774, 4.427)
Ds 4.146 (0.534) 0.909 0.161 3.885 (3.830, 4.462)
Dd 4.066 (0.534) 0.899 0.170 4.177 (3.733, 4.399)
Probe
D∆ 0.080 (0.037) 0.512 0.026 32.50 (0.029, 0.131)
Dm 4.140 (0.531) 0.980 0.075 1.815 (3.992, 4.287)
Ds 4.197 (0.519) 0.970 0.090 2.141 (4.021, 4.373)
Dd 4.109 (0.514) 0.975 0.081 1.980 (3.950, 4.269)
Freq.
D∆ 0.088 (0.036) 0.255 0.031 35.26 (0.027, 0.149)
Dm 4.176 (0.487) 0.967 0.088 2.118 (4.003, 4.349)
Ds 4.209 (0.495) 0.973 0.081 1.934 (4.049, 4.368)
Dd 4.143 (0.483) 0.966 0.089 2.148 (3.969, 4.318)
Location
D∆ 0.065 (0.041) 0.617 0.025 38.62 (0.016, 0.115)
Dm 4.166 (0.488) 0.994 0.038 0.908 (4.092, 4.240)
Ds 4.219 (0.497) 0.986 0.059 1.393 (4.103, 4.334)
Dd 4.145 (0.491) 0.995 0.035 0.838 (4.077, 4.213)
Display
D∆ 0.073 (0.051) 0.596 0.032 43.83 (0.010, 0.136)
Mean (SD) diameters for each ultrasound setting. Absolute difference (D), coefficients of variation (CV), intra-class cor-
relation coefficient (ICC), and standard error of measurement (SEM) plus confidence intervals (CI) are shown.
-0.20
-0.15
-0.10
-0.05
0.00
0.05
0.10
0.15
0.20
2.9 3.43.9 4.4 4.9
Average of T1 & T3 (m m )
T3 - T1 (mm )
Mean
- 1.96 S D
+ 1 .9 6
Figure 3. Bland-Altman plot of diameter difference values
(Trial 3 - Trial 1) on average diameter values ((Trial 1 + Trial
3)/2)). The mean difference, upper boundary ((mean + (SD of
mean × 1.96)), and lower boundary ((mean – (SD of mean ×
1.96)) are shown.
D∆ than for single diameters. Bland-Altman plots (not
shown) indicate bias towards larger D∆ for the 539 probe
compared to the other two probes.
4. DISCUSSION
This study shows that arterial diameters can be reliably
-0 .12
-0 .08
-0 .04
0.00
0.04
0.08
0.12
0.00 0.04 0.08 0.12
Avg. 11mhz & 8mhz ( m m )
Avg. 8m hz - 11m hz ( m m)
Mean
- 1.96 SD
+ 1 .96
Figure 4. Bland-Altman plot of distension difference values (8
MHz – 11 MHz) on average distension values ((11 MHz + 8
MHz)/2)). The mean difference, upper boundary ((mean + (SD
of mean × 1.9.6)), and lower boundary ((mean – (SD of mean
× 1.96)) are shown.
measured within-session, but that measurement error for
arterial distention calculation is notably higher. Also, it
was found that variations in ultrasound probe selection
and optimization settings contribute to some measure-
ment bias, though with little impact on the reliability of
diameter measurements. However, variations in ultra-
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L. Stoner et al. / Open Journal of Clinical Diagnostics 1 (2011) 15-21 20
-0 .6
-0 .4
-0 .2
0.0
0.2
0.4
0.6
2.9 3.4 3.94.4 4.9
Avg. of LA39 & 546 ( m m )
Avg. 54 6 - LA39 (mm)
Mean
- 1.96 SD
+ 1.96 SD
Figure 5. Bland-Altman plot of diameter difference values
(546 – LA39) on average values ((LA39 + 546)/2)). The mean
difference, upper boundary ((mean + (SD of mean × 1.9.6)),
and lower boundary ((mean – (SD of mean × 1.96)) are shown.
sound probe selection and optimization settings notably
impact the reliability of arterial distention measure-
ments.
4.1. Probe Selection and Frequency Settings
Across ultrasound settings we found that probe selection
had the greatest impact on reliability for both diameter
and arterial distention measurements. The lower fre-
quency bandwidth probe (546, 6.6 MHz) resulted in bias
towards smaller diameter and distention measurements.
Decreasing the frequency of the LA39 probe also re-
sulted in bias towards smaller diameter values, although
this did not affect distension.
Probe selection and frequency settings may be de-
pendent on the population sample, i.e., subjects with
higher subcutaneous fat will require a probe that oper-
ates at a lower frequency bandwidth. Probe selection
may also be dependent on the artery being assessed, i.e.,
deeper arteries will require a lower frequency bandwidth.
Furthermore, during the course of an intervention study,
optimal probe selection may be dependent on body
compositional changes. This study shows that probe
selection needs to be standardized for a given subject if
repeated measures are to be made. Furthermore, diag-
nostic meaning for population comparisons may be in-
fluenced if different probes are used to compare popula-
tions.
4.2. Ultrasound Imaging Mode Selection and
Imaging Field
Another important finding of this study pertains to the
imaging display, i.e., B-mode vs. PD-mode (duplex). We
found that PD-mode results in marginally smaller di-
ameter and distention values. However, altering the im-
aging display did not affect the reliability of these meas-
urements. We found that changing the location from a
near-field to far-field did not notably impact reliability,
but did result in progressive bias towards smaller di-
ameters. The imaging display selected will be dependent
on whether simultaneous blood velocity measurements
are required. Reliable measurements can be made when
imaging in PD-mode, however if repeated measures are
to be made, then it is advisable that the imaging display
remain constant. It is also advisable that image focus is
maintained central to the ultrasound field.
5. CONCLUSIONS
Ultrasound can reliably measure arterial diameters and
distension for the duration of a given test session. How-
ever, alterations to probe selection and optimization set-
tings – particularly probe selection – can have a signify-
cant impact on measurement precision. Although reli-
ability is often more important than absolute accuracy
for serial exams, measurement differences due to varia-
tions in instrument settings may, nevertheless, be inter-
preted as having substantive diagnostic meaning. It is
therefore recommended that ultrasound probe selection
and optimization settings are standardized for repeated
measurements, and preferably across subjects.
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