Perspectives in Medicine (2012) 1, 170176

Bartels E, Bartels S, Poppert H (Editors):

New Trends in Neurosonology and Cerebral Hemodynamics an Update.
Perspectives in Medicine (2012) 1, 170176

journal homepage: www.elsevier.com/locate/permed

Comparative in vivo and in vitro postmortem

ultrasound assessment of intima-media thickness
with additional histological analysis in human carotid
arteries
Szabolcs Farkas a,, Sndor Molnr b, Katalin Nagy a, Tibor Hortobgyi a,c,1,
Lszl Csiba a,1

a
Department of Neurology, University of Debrecen Medical and Health Science Center, 98 Nagyerdei Street, Debrecen H-4012,
Hungary
b
Department of Neurology, Sopron Elizabeth Hospital, 15 Gyori Street, Sopron H-9400, Hungary
c
Department of Pathology, University of Debrecen Medical and Health Science Center, 98 Nagyerdei Street, Debrecen H-4012,
Hungary

KEYWORDS Summary The present study aims to validate the technique of in vitro ultrasonography (US)
IMT; by comparative analysis of premortem intimamedia thickness (IMT), postmortem IMT and
In vitro average wall thickness. In vivo common carotid artery (CCA) IMT was measured bilaterally in
ultrasonography; 25 patients at 30 mm proximal from the ow divider. After autopsy in vitro US was performed
In vivo and postmortem IMT was measured at the same level. Snap frozen arterial specimens were
ultrasonography; processed for average wall thickness determination and for histology. High degree of correlation
Common carotid was found between in vivo IMT, in vitro IMT and average wall thickness. Our results demonstrate:
artery; (1) in vitro US is a reliable and reproducible tool for the examination of autopsied arterial
Snap freezing; specimens to obtain valuable information about vascular wall properties and to identify the
Comparative analysis optimal vascular segment for tissue sampling; (2) snap freezing and cryosectioning of in toto
excised arterial specimens is recommended for comparative histologicalUS studies.
2012 Elsevier GmbH.Open access under CC BY-NC-ND license.

Introduction

Atherosclerosis is a generalized arterial disease that starts

Corresponding author at: University of Debrecen-Medical and
Health Science Center, Department of Neurology, Mricz Zsigmond
Street, no. 22, H-4032 Debrecen, Hungary.
Tel.: +36 52 411 717x55986; fax: +36 52 453 590.
E-mail address: endreszabi@gmail.com (S. Farkas).
1 L.C. and T.H. are the senior authors.
decades before the onset of clinical symptoms, such as
angina pectoris, myocardial infarction or stroke [13].
Atherosclerosis in main arteries begins with the enlarge-
ment of the vascular lumen and size [47]. Necropsy studies
conrmed the premorbid, age-related increase of intimal
and medial thickness [79]. It has been suggested that
early increase in intimamedia thickness (IMT) reects

2211-968X 2012 Elsevier GmbH. Open access under CC BY-NC-ND license.

doi:10.1016/j.permed.2012.02.050
In vivo and in vitro carotid ultrasound: comparative analysis 171

adaptation to elevated intravascular shear stress whereas

Table 1 Summary of patients data.
increased IMT with US detectable atherosclerotic plaque is
associated with end-organ disease [1012]. Risk factors, Number of patients n = 25
including age, gender, diabetes mellitus, blood pressure, Age (years) 76.4 11.5
smoking, elevated serum low-density lipoprotein choles- Gender (male:female) 9:16
terol, homocystein levels and chlamydia infestation of the
Risk factors Hypertension (No; 20 (80%)
arterial wall are also associated with larger diameter and
%)
IMT of the extracranial carotid arteries [4,13].
Diabetes mellitus 8 (32%)
In vivo intimamedia thickness (IMT) measured non-
(No; %)
invasively by high resolution B-mode ultrasonography is
Diagnosis at admission Cerebral 14
considered as a valid and reliable indicator of the local
infarction (No)
and generalized expansion of subclinical, later, clinical
Cerebral 3
atherosclerosis [8]. IMT is dened as the distance between
hemorrhage (No)
blood-intima and media-adventitia interfaces of arterial
Others (No) 8
wall [14]. Most often it is measured at the common carotid
artery (CCA), because high measurement precision can Cause of death Raised intracranial 5
be obtained in this artery. These are made over a dis- pressure (No)
tance of 1 cm at levels 12 cm proximal to the bifurcation; Pneumonia (No) 6
a mean value for the selected area is obtained using Pulmonary 5
automated wall-tracking software [5,15,16]. Nevertheless, embolism (No)
in vivo carotid IMT determination aims primarily the far arte- Cardiac failure 8
rial wall (the side further to the US transducer) since an (No)
accurate measurement of the near wall IMT is extremely Sepsis (No) 1
difcult and requires a high level of technical expertise Data are means SEM.
[17,18]. Moreover, meta-analysis revealed that circumfer-
ential scanning of the carotid artery and calculation of
the mean maximum carotid IMT provides a more accurate
for postmortem vascular wall investigation, and to examine
measurement of carotid atherosclerosis [19]. In addition,
the applicability of snap freezing histotechnique on utilized
anatomy, motion artifacts or ultrasound equipment can
vascular specimens.
also inuence in vivo IMT determination [2023]. A vari-
ety of non-invasive imaging techniques and softwares have
been used to improve in vivo IMT determination and to
Patient selection, materials and methods
increase the reliability of IMT as marker of atherosclero-
sis [18,20,2225]. However, these IMT measuring methods
have not been validated yet and a quick and reliable method Patients
for initial in vitro testing of new techniques and softwares,
apart from the widely used in vivo US, is also needed. The Comparisons between ultrasound and postmortem ndings
present study addresses these deciencies. were performed in 25 patients. Table 1 contains general
It has been suggested that changes in intensity of shear data about patients. The study was approved by the local
stress inuence the arterial wall responses from less to more Ethics Committee and informed consent was obtained from
proliferative phenotypes, which may underlie the differ- the relatives of each examined individual.
ences in genetic effects on CCA IMT and bifurcation IMT [26].
Further research is required to clarify genetic effects and
local gene expression patterns, which could inuence patho- In vivo ultrasonography
logical processes in arterial walls and hence the arterial IMT.
Therefore, a better knowledge of gene-IMT associations, SONOS 4500 ultrasound system (Agilent, Andover, MA, USA)
i.e. taking into consideration US IMT measurements in the with a 311-MHz linear transducer was used for in vivo and
context of gene expression prole data could improve the in vitro ultrasonography. In vivo IMT measurements were
accuracy and reliability in prediction of the progression of performed in a longitudinal B-mode projection while the
atherosclerotic vascular disease. patient was in a supine position. IMT was determined as
It is accepted that freezing of excised tissues could result the distance from the leading edge of the rst echogenic
in alteration of microanatomic structure especially because line to the leading edge of the second echogenic line of the
of ice crystal formation [27]. On the other hand, histological double line pattern of the far artery wall (Fig. 2). Three
preparation of arterial sections affects vascular and plaque measurements along a 23-mm portion of the vessel were
dimensions [2831]. Another aspect of our study was to performed and were averaged. IMT measurements site on
reveal whether snap freezing of arterial samples inuences the CCA were localized by the distance of 30 mm from tip
histological preparation of arterial specimens. of the ow divider. This landmark enabled us to reconstruct
In the present study we compared in vivo IMT with in vitro the position of the in vivo IMT measurement later during
US measured IMT and average wall thickness. Finally, his- the postmortem IMT determination. Wall thickening over
tological processing of selected frozen arterial specimens 2 mm was determined as plaque and excluded from further
was also performed. We aimed to validate in vitro US as evaluation, which resulted in an important screening of the
alternative method, if in vivo US data were not available, postmortem usable arterial specimens.
172 S. Farkas et al.

Figure 1 Processing of autopsied arterial specimens. Autop-

sied carotid arteries were lled with histological embedding
material (Cryochrome Blue) (image A) and in vitro ultrasonog-
raphy (US) was performed (image B). This was followed by
snap freezing of the lled specimens and, subsequently, 3 mm
thick sections were cut (image C) for performing planimetric
analysis and histological processing of the samples: CCA, com-
mon carotid artery; ICA, internal carotid artery; ECA, external
carotid artery.

Preparation of the arteries for in vitro IMT and

average wall thickness determination

Within 24 h after death, 4 cm of common carotid arteries

(CCA) and 4 cm of the proximal segments of internal- and
external carotid arteries (ICA and ECA) were removed in
toto from both sides. The native vessels were lled with
histological embedding material (Cryochrome Blue; Thermo
Shandon, Pittsburgh, PA, USA) and a constant pressure of
100 mmHg was adjusted (Fig. 1). The presence of ICA and
ECA helped us to identify the anatomical position during
the insonation to visualize precisely the far and near arte-
rial. Subsequently, in vitro IMT was measured in 34 CCAs
as described upper using ultrasound gel during the direct
contact between transducer and prepared arterial speci-
mens. In vitro measurements were compared with in vivo
IMT values (Fig. 2). A thread has been xed at 3 cm dis-
tance from tip of the ow divider in order to mark the
exact location where in vitro IMT measurements were per-
formed. Afterwards, lled specimens were frozen at 20 C
in a box containing embedding material, and subsequently,
Figure 2 Comparison of common carotid artery (CCA) IMT
measured by in vivo ultrasonography (US) (image A), in vitro
US (image B) and histological method (image C). Ultrasono-
graphic IMT is dened as the distance between the leading edge
of the rst echogenic line and the leading edge of the second
echogenic line (A and B) and it was measured at 30 mm dis-
tance proximal from the bifurcation of CCA. Histological image
(C) shows arterial wall in 40-fold magnication with good tissue
preservation. Arrows indicate the lumen-intima and media-
adventitia interfaces.
cut into 3 mm thick slices (Fig. 1) as described previously
[31,32]. Consecutive slices were photographed with a high-
resolution (3040 2016 pixels) digital camera (FinePix S1
Pro; Fuji Photo Film Co., Tokyo, Japan) for digitization and
planimetric analysis of the sections. Six to eight slices per
In vivo and in vitro carotid ultrasound: comparative analysis
vessel were evaluated. A calibration bar was also digitized
with each individual sample to determine the magnication
of the system and to convert the pixel values into millime-
ters.
Calculations
The measured parameters (IMT, average wall thickness)
were expressed in millimeters. Mean values of the measured
in vivo IMT, in vitro IMT and average wall thickness were
calculated.
Mean differences between in vivo and in vitro IMT were
expressed in millimeters and percents according to the fol-
lowing formulas:
IMT difference (mm) = in vitro IMT in vivo IMT;
IMT difference(%) = (in vitro IMT in vivo IMT)/in vitro IMT
100, respectively
Vessel circumference and lumen circumference on the
digitized 3 mm thick arterial sections were measured with
free available image analyzer software of the National
Institute of Health and mean values were calculated. Sub-
sequently, average wall thickness was determined based on
the following formula: average wall thickness (mm) = (vessel
circumference lumen circumference)/2.
Histological processing
CCA specimens were processed for histology. Three mil-
limeter thick frozen arterial slices prepared as described
above and marked by the thread at the level of in vitro IMT
measurements were used. Afterwards, transverse sections
(20 m) of the marked slices were cut by cryomicrotome
(Leica, CM 1850, Stockholm, Sweden) and were stained
with hematoxylin & eosin (H&E) and VerhoeffVan Gieson
[33,34]. Sections with articially damaged intima and/or
media at the site of the measurement were excluded.
Statistical analysis
Concordance analysis was performed between in vivo IMT
and in vitro IMT measurements. Furthermore, BlandAltman
plots were applied to illustrate the agreement between
in vitro and in vivo IMT measurements [20]. Linear regres-
sion analysis was preformed to correlate in vivo IMT, in vitro
IMT and average wall thickness.
Results
In the present study we have compared postmortem IMT
determination with in vivo IMT and average wall thickness.
Furthermore, histological processing of selected snap frozen
arterial specimens was performed.
173
Table 2 Comparison of IMT measurements on common
carotid artery.
IMT Means SD Range
In vivo (mm) 0.93 0.10 0.671.33
In vitro (mm) 0.97 0.14 0.611.68
Differencea (mm) 0.046 0.173 to 0.333
Differenceb (%) 3.8 20 to 25
Data are means SEM, n = 34.
a Difference (mm) = in vitro in vivo.
b Difference (%) = (in vitro in vivo)/in vitro 100.
In vitro and in vivo ultrasonographic IMT
measurements, histology
In vivo and in vitro IMT measurements were compared in
n = 34 CCA specimens. Fig. 2 presents in vivo and in vitro
IMT measurements as well as histological image of H&E
stained snap frozen arterial section. Results are summa-
rized in Table 2. According to our results the mean IMT was
0.93 0.12 mm by in vivo US and 0.97 0.18 mm by in vitro
ultrasound. The concordance between the two groups was
signicant: concordance coefcient RC = 0.545, p < 0.0001,
95% condence interval 0.3360.755. Concordance analysis
and BlandAltman plots for both parameters are shown in
Fig. 3.
Ultrasound and gross pathology
Average wall thicknesses were calculated in case of n = 34
CCA specimens. Both in vitro and in vivo IMT values cor-
related well with average wall thicknesses measured at
the corresponding postmortem samples (r = 0.76, R2 = 0.571;
r = 0.57, R2 = 0.328, respectively). Fig. 3 presents the scat-
ter plots and regression equations for average wall thickness
related to in vivo and in vitro US IMT.
Discussion
In this study we analyzed the degree of correlation between
in vivo IMT, in vitro IMT, and the average wall thickness
examined in human common carotid arteries. We found
signicant concordance between in vivo and in vitro US
determined IMT. Both corresponded well with the calculated
average wall thickness. Following the in vitro tissue pro-
cessing tissue preservation, shrinkage and overall suitability
for microscopic analysis was assessed on stained histological
sections from snap-frozen arterial segments. The applica-
bility of in vitro US on autopsied vascular specimens has
been demonstrated; and conrmed that postmortem IMT
measured by in vitro US can be used as reliably as in vivo
IMT.
It is well known the fact that through freezing water
expands and forms ice crystals. This process can result in
freezing artifacts and tissue damage, which, however, can
be prevented by reduced freezing time [27]. Formalin x-
ation, dehydration in ethanol or other agents and parafn
embedding during processing could result in up to a 3040%
tissue shrinkage, changing vascular dimensions and causing
174 S. Farkas et al.

Figure 3 Concordance analysis (A) and BlandAltman plots (B) of the in vivo and in vitro ultrasonographic (US) measured CCA
IMT; scatter plots of in vivo US IMT and average wall thickness at CCA (C) and scatter plots of in vitro US IMT and average wall
thickness at CCA (D). R2 and regression equations are included. IMT measurements and average wall thickness values are expressed
in millimeters: IMT, intimamedia thickness; CCA, common carotid artery.

discrepancy between US and histological IMT measurements
[2831]. CCA IMT values obtained with in vitro US and
follow-up histological determination showed good agree-
ment (data not shown). However, due to the low number
of available specimens for histological processing statisti-
cal analysis between in vitro and microscopic IMT was not
performed. In this study we presented that in vitro tissue
processing by snap freezing results in low extent of tissue
shrinkage and minimal change in vascular wall properties.
Therefore frozen postmortem artery sections are compara-
ble with data derived from US methods both in vivo and
in vitro and frozen sections are suitable for histologicalUS
comparative analytical studies.
Despite the fact that carotid IMT is a well established sur-
rogate marker for clinical events, in vivo US measured wall
thickness has a variability caused by anatomy, ultrasound
equipment, angle of insonation, attenuation of US by neck
muscles, motion artifacts (swallowing, arterial pulsation and
breathing) and examiner skills [2023]. Furthermore, in
vivo US investigates mainly the IMT of the far vessel wall,
however, atherosclerotical processes and IMT changes are
also present in other parts of vascular wall, therefore, a
circumferential wall thickness determination is more reli-
able. In addition, there is a need for new in vivo imaging
methods providing a detailed view of the arterial tree and
vessel wall [17]. Magnetic resonance imaging (MRI) provid-
ing detailed cross-sectional images of all sides of carotid
artery wall and three-dimensional motion sensitized seg-
mented steady-state black-blood gradient echo technique
(3D MSDS) with rapid artifact-free overview imaging of the
carotid wall are very promising techniques [21,24]. Finally,
it has been suggested that computer-aided measuring tech-
niques could result in increased accuracy and reduction of
operator dependent subjectivity of IMT determination [23].
In our present study we have demonstrated that measuring
IMT in postmortem arterial specimens by US is a reliable and
reproducible method, which could be used for US standard-
ization in subsequent studies. Hence, in vitro US measured
IMT could be used to develop, improve, compare or validate
new imaging techniques (e.g. fast three dimensional imag-
ing techniques), automated IMT measurement algorithms,
or new softwares for ultrasound methods.
Carotid IMT is strongly determined by genetic factors act-
ing independently of traditional cardiovascular risk factors
[35,36]. A heritability range of 2040% has been estimated
by studies in unselected subjects, twins, and people with
type II diabetes [3640]. Genes related to hemostasis,
lipid and lipoprotein levels, extracellular matrix remodel-
ing, antioxidation, reninangiotensin system, endothelium
function, inammation have been associated with carotid
IMT changes [34,41]. On the other hand, laminar ow and
oscillatory shear stress trigger diverse local endothelial
responses and altered gene expressions and result in an
atherogenic phenotype [26,4244] which may vary in dif-
ferent carotid segments with a possible impact on IMT. Our
results implicate that in vitro US including IMT provide valu-
able information about autopsied arterial specimens. These,
afterwards, can be stored and made available in tissue banks
In vivo and in vitro carotid ultrasound: comparative analysis
for a wide range of -omics investigations. In addition, in
vitro US of arterial specimens could serve as a guide to
identify the most appropriate region of an intact autopsied
vascular tissue for histological sampling.
Furthermore, Liao et al. (2008) applied the gene risk
score (GRS) to estimate a cumulative effect of genes signif-
icantly associated with IMT and emphasize the importance
of future gene-IMT association studies on different popula-
tions. The use of the GRS may simplify an assessment of
multiple gene effects in complex diseases and may provide
a better estimate of individual susceptibility to atheroscle-
rosis [26]. The accuracy and utility of GRS can possibly
be improved by including an US artifact free postmortem
IMT measurements of different parts of arterial wall (e.g.
far wall, near wall, etc.). GRS combined with IMT could
improve the precision and reliability of prognosis determi-
nation models for a complex disease like atherosclerosis.
In the present study we measured arterial IMT apply-
ing in vitro US and compared it to in vivo determined
IMT, histological IMT and average arterial wall thickness.
We demonstrate that for microscopic IMT determination
purposes cutting and processing frozen arterial sections
after in vitro US is a suitable histological technique which
has advantages compared to use of formalin xed paraf-
n embedded (FFPE) slides. Our data conrm that frozen
sections from autopsied arteries are suitable for both histo-
logical IMT determination and UShistological comparative
analysis.
We demonstrate that postmortem in vitro US is a reli-
able and reproducible technique for detection of arterial
wall changes as alternative method of its in vivo ana-
logue. In addition, validated in vitro US is a reliable tool
to identify, without plaque manipulation, the vascular seg-
ment for tissue sampling. In particular, it is as suitable for
IMT determination as in vivo US, without the methodologi-
cal/technical/ethical limitations of in vivo human studies.
Standardized in vitro US measured IMT provides basis for the
development and validation of novel non-invasive imaging
techniques to study vessel wall abnormalities.
In conclusion, in vitro US can be widely used in vascu-
lar research with the potential of correlative morphological,
genetic, biochemical and imaging to study complex vascular
diseases such as arteriosclerosis.
Acknowledgements
Drs Lszl Kardos and Katalin Hegeds are thankfully
acknowledged for statistical and general advice. The authors
express their gratitude to Katalin Nagy for the outstanding
technical assistance.
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