Optical Coherence Tomography for Investigation

Optical Coherence Tomography for Investigation of the Pancreatico-
Biliary System: Still Experimental?
Pier Alberto Testoni, Benedetto Mangiavillano, Alberto Mariani
Department of Gastroenterology and Gastrointestinal Endoscopy, IRCCS Vita-Salute San Raffaele
University, San Raffaele Hospital Scientific Institute. Milan, Italy
Optical coherence tomography (OCT) is an
optical imaging modality introduced in 1991
[1] that performs high-resolution, cross-
sectional, subsurface tomographic imaging of
the internal microstructure in materials and
biologic systems by measuring backscattered
or backreflected infrared light.
The physical principle of OCT is similar to
that of B-mode ultrasound imaging, except
that the intensity of infrared light, rather than
sound waves, is measured. Wavelenghts of
the infrared light used in OCT are one to two
orders of magnitude higher than ultrasound
wavelenght, so OCT technology can yield a
lateral and axial spatial resolution of about 10
micron, which is 10- to 25-fold better than
that of available high-frequency ultrasound
imaging. The spatial resolution of OCT
images is nearly equivalent to that of
histologic sections. The depth of penetration
of OCT imaging is approximately 1-3 mm,
depending upon tissue structure, depth of
focus of the probe used, and pressure applied
to the tissue surface. Although the progressive
increase in ultrasound resolution is
accompanied by a corresponding decease in
depth of penetration, a similar trade-off
between resolution and depth of penetration
does not occur in OCT imaging. In contrast to
magnification endoscopy, OCT has depth.
Several in vitro studies demonstrated the
feasibility of OCT in the gastrointestinal (GI)
tract: in these studies the GI tract wall was
identified as a multiple layer structure
chraracterized by a sequence of hyper- and
hypo-reflective layers, with a variable
homogenicity of the back-scattered signal [2,
3, 4]. Neoplastic and normal tissue also
showed different light backscattering patterns
[5]. However, the optical properties of
nonliving tissues are different from tissue in
vivo.
Subsequent studies were therefore performed
in ex-vivo tissue specimens and aimed at
comparing OCT imaging with histology, to
assess the realiability of the OCT technique to
identify and recognize the GI wall structure.
OCT was shown to clearly differentiate the
layers’ structure of the GI wall [6].
In the last decade, OCT technology has
evolved from an experimental laboratory tool
to a new diagnostic imaging modality with a
wide spectrum of clinical applications in
medical
practice,
including
the
gastrointestinal tract and pancreatico-biliary
ductal system.
Technical OCT Principles of Operation
OCT devices use a low-power infrared light
with a wavelenght ranging from 750 to 1300
nm in which the only limiting factor is the
scattering of light. Scattering occurs when the
light interacts with tissue surface and the
image formation depends upon the difference
in optical backscattering properties of the
tissue.
OCT images are generated from measuring
the echo time delay and the intensity of

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backscattered light. Because the velocity of
light is extremely high, optical echoes cannot
be measured by direct electronic detection,
but by means of a low-coherence
interferometry that measures the interference
of two incident light beams that are derived
from a single source of low-coherence light.
Low-coherence light can be generated by
compact superluminescent semiconductor
diodes or other sources, such as solid-state
lasers.
In the low-coherence interferometry the light
reflected or backscattered from inside the
specimen is measured by correlating with
light that has traveled a known reference path.
One interferometer arm contains a modular
probe that focuses and scans the light onto the
tissue sample, also collecting the
backscattered light; the second interferometer
arm is a reference path with a translating
mirror or scanning delay line [7].
Lights reflected from the specimen and
reference beam are combined at a detector,
and the interference between the two beams is
measured. Optical interference between the
light from the sample and reference path
occurs only when the distance traveled by the
light in both paths matches to within the
coherence length of the light [8].
The coherence length of the light source,
which is inversely proportional to its spectral
bandwidth, determines the axial or depth
resolution of OCT: the shorter the coherence
length of the light source, the better is the in-
depth spatial resolution. The spatial width of
the scanning beam determines the lateral or
transverse resolution. The image penetration
depth is determined by the absorption and
scattering properties of the sample surface.
In OCT, two-dimensional cross-sectional
images of tissue microstructure are
constructed by scanning the optical beam and
performing multiple axial measurements of
backscattered light at different transverse
positions. The resulting data set is a two-
dimensional array that represents the
displayed as a grey-scale or false-color image.
Three types of scanning patterns are available
for OCT imaging: radial [9, 10, 11],
longitudinal [12, 13], and transverse [14]. The
radial-scan probe directs the OCT beam
radially, giving images that are displayed in a
"radar-like", circular plot. Radial scanning
can easily image large areas of tissue by
moving the probe forth and back over the
tissue surface and has the highest definition
when the probe is inserted within a small
diameter lumen, because the OCT images
become progressively coarser when a large-
diameter lumen is scanned, due to the
progressive increase of pixel spacing with
increasing the distance between the probe and
the tissue. The linear and transverse probes
scan the longitudinal and transverse positions
of the OCT beam at a fixed angle, generating
rectangular images of longitudinal and
transverse planes at a given angle with respect
to the probe. Linear scanning has the
advantage that pixel spacing in the transverse
direction is uniform and can better image a
definite area of the scanned tissue, expecially
in presence of large-diameter and non-circular
lumens, where maintaining constant distance
from the probe to the surface over the entire
circumferential scan may be impossible.
Transverse scanning modality provides a
better depth of field. Depth of field is the
range of distances from the probe over which
optimal resolution of scaninng can be
obtained; current OCT scans permit imaging
depths of up to 2-3 mm in tissues, by using
probes with different focuses.
OCT Technique for the Pancreatico-
Biliary Ductal System Investigation
OCT imaging from the GI tract can be done in
humans by using narrow-diameter, catheter-
based probes [15]. The probe is deteachable
from the OCT main unit, making it
reprocessable between procedures.
The probe can be inserted through the
accessory channel of a side-view endoscope,
inside a standard transparent ERCP catheter,
for investigating the pancreatico-biliary ductal
system (Figure 1). The possibility to introduce
the OCT probe into the pancreatico-biliary
ductal system permits to investigate in detail
the epithelial layers of the ductal system and
sphincter of Oddi. OCT scanning can be done
either keeping the OCT probe in the ERCP

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catherer or leaving it in the duct outside the
catheter (Figure 2).
From a clinical point of view, OCT imaging
of the pancreatic and biliary ductal system
could improve the diagnostic accuracy for
ductal epithelial changes and the differential
diagnosis between neoplastic and non-
neoplastic lesions, since in several conditions
X-ray morphology obtained by ERCP and
other imaging techniques may be non-
diagnostic, and the sensitivity of intraductal
brush cytology during ERCP procedures is
highly variable.
In our studies a near-focus OCT probe
(Pentax, Lightlab Imaging, Westford, MA,
USA) was used, with a penetration depth of
about 1 mm and a resolution of approximately
10 μm. The probe operates at 1.2-1.4 μm
center wavelength (nominal value: 1.3 μm),
with a scan frequency ranging from 1,000 to
4,000 kHz (nominal value: 3,125 kHz). Radial
and longitudinal scanning resolutions have an
operating range in tissue of 15-20 μm
(nominal value: 18 μm), and 21-27 μm
(nominal value: 24 μm) respectively. Infrared
light is delivered to the imaging site through a
single optical fiber 0.006-inch diameter. OCT
probe is assembled in a catheter with an outer
diameter of 1.2 mm, The catheter-based probe
consists of a rotating probe encased in a
transparent outer sheath which remains
stationary while the rotating probe has a pull-
back movement of 1 mm/second, with an
acquisition rate of 10 frames per second. By
this technique a segment of tissue 5.5 cm long
can be filmed over a 55-second period.
Experimental Investigation by OCT of the
Pancreatico-Biliary Ductal System
Normal Pancreatico-Biliary Ductal System
Visualization of the normal epithelium of the
main pancreatic duct has been obtained post-
mortem [16] and ex-vivo in humans [17, 18],
while in-vivo it comes from one study in
animals [19]. Normal biliary ductal system
has been investigated in humans, ex vivo in a
study [17], post-mortem [16], and in-vivo in
animals and human being [19, 20, 21].
Tearney et al. in 1998 [16] published the first
data in literature about the use of the OCT in
the pancreato-biliary system, in a post-
mortem study in human beings. OCT images
of the common bile duct (CBD) wall were
able to identify the layered structure and
resolve the submucosa-muscularis and
muscularis-adventitia boundaries. Different-
iation of the submucosa, muscularis and
adventitial layers was made possible by
visualization of the different back-scattering
characteristics within each layer. The
adventitial layer seemed to have a lower and
more irregular back-scattering intensity than
the submucosa or muscularis. This irregular
back-scattering pattern is most likely due to
the presence of adipose tissue into the
adventitial layer. High-resolution OCT
imaging enabled tissue microstructure, such
as secretions within individual glands, and
cross-sectional imaging of islets Langherans
cells to be visualized. The pancreatic duct,
Figure 2. OCT probe inside (a.) and outside (b.) the
ERCP catheter.
Figure 1. The duodenoscope with OCT mid-probe
inside the ERCP catheter.

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moreover, appeared as a highly back-
scattering band near the lumen of the tissue.
Pancreatic stroma was seen beneath the
pancreatic duct.
Recently Singh et al. [19] published a study
in-vivo in animals (dogs) on the OCT imaging
of the pancreatic and biliary ductal system.
OCT was able to identify the entire wall of
the bile duct and some of the surrounding
fibrous tissue but could not identify adjacent
structures such as blood vessels. A thin, low-
scattering, superficial layer could be
discerned on the majority of the images,
which was thought to represent the cuboidal
epithelium. Nuclei or subcellular structures
could not be identified. The lamina propria
appeared to be a highly reflecting layer
underneath the mucosal surface. The dense
connective tissue underlying the lamina
propria was imaged as linear layers of
differing intensities. Peribiliary glands were
seen as large open spaces with a single layer,
signal-poor, epithelium. The pancreatic duct
in dogs had a flat mucosal layer composed of
cuboidal epithelium. OCT was able to image
the wall of the pancreatic duct but not the
surrounding parenchyma. The pancreatic duct
on OCT images was homogeneous and
moderately reflective.
In a study by our group [17], OCT imaging of
main pancreatic duct, common bile duct and
sphincter of Oddi normal structure has been
shown to be able to provide features that were
similar to those observed in the corresponding
histological specimens in 80% of sections; the
agreement between OCT and histology in the
definition of normal wall was good (81.8%).
OCT images identified three differentiated
layers up to a depth of about 1 mm. From the
surface of the duct, it was possible to
recognize an inner hypo-reflective layer
corresponding to the single layer of epithelial
cells close to the lumen, an intermediate
homogeneous
hyper-reflective
layer
corresponding to the fibro-muscular layer
surrounding the epithelium, and an outer, less
definite, hypo-reflective layer corresponding
to the smooth muscular structure within a
connective tissue in the common bile duct and
at the level of the sphincter of Oddi, and
connective-acinar structure in the main
pancreatic duct (Figures 3, 4, 5).
The inner, hypo-reflective layer showed a
mean thickness of 0.05 mm (range 0.04-0.08
mm) and a homogeneneous back-scattering of
Figure 3. Magnification of an OCT image from the
normal common bile duct wall, compared with
histology. From the surface of the duct, up to a depth of
1 mm, the following layers are recognizable: a) the
single layer of epithelial cells, approximately 0.04-0.06
mm thick, visible as a superficial, hypo-reflective band;
b) the connective-muscular layer surrounding the
epithelium, visible as a hyper-reflective layer
approximately 0.34-0.48 mm thick; c) the connective
layer visible as a hypo-reflective layer with
longitudinal relatively hyper-reflective strips (smooth
muscle fibers).
Figure 4. Magnification of an OCT image from the
normal sphincter of Oddi wall, compared with
histology. From the surface of the duct, up to a depth of
1 mm, the following layers are recognizable: a) the
single layer of epithelial cells, approximately 0.04-0.08
mm thick, visible as a superficial, hypo-reflective band;
b) the connective-muscular layer surrounding the
epithelium, visible as a hyper-reflective layer
approximately 0.23-0.37 mm thick; c) the connective
layer visible as a hypo-reflective layer with
longitudinal relatively hyper-reflective strips (smooth
muscle fibers). Within the intermediate and outer layer
vessels are also recognizable, visualized as non-
reflecting areas surrounded by an hypo-reflective
endothelium. Margins between the intermediate and
outer layer are poorly recognizable, due to the irregular
distribution of connective and muscular structure.

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the signal in all the imaged sites; thickness,
surface regularity, and reflectance degree of
this layer did not substantially differ in the
common bile duct, main pancreatic duct, and
sphincter of Oddi. The intermediate layer
showed a mean thickness of 0.41 mm (range:
0.34-0.48 mm) in the common bile duct, 0.42
mm (range: 0.36-0.56 mm) in the main
pancreatic duct, and 0.29 mm (range: 0.23-
0.37 mm) in the sphincter of Oddi. The layer
thickness was substantially similar in the
ducts, while appeared reduced by 25% at the
level of the sphincter of Oddi. The layer
appeared hyper-reflective when compared
with the inner and outer layers and the
reflectance degree did not change in all the
imaged sites. Within the context of the
intermediate layer, tiny, multiple, non-
reflective areas were recognizable in the main
pancreatic duct and at the level of sphincter of
Oddi. The outer layer appeared recognizable
until a depth of about 1 mm from the lumen
and was hypo-reflective in all the imaged
sites. Multiple, hyper-reflective, longitudinal
strips were recognizable at the level of
common bile duct and sphincter of Oddi.
These longitudinal strips were more
pronounced and hyper-reflective in the
sphincter of Oddi, so the layer appeared at
this level less hypo-reflective than in the
common bile duct.
The three different layers showed a linear,
regular surface and each layer had a
homogeneous back-scattered signal in every
frame; however, the differentiation between
the intermediate and outer layer appeared
more difficult than between the inner and
intermediate layer. The thickness of the inner
and intermediate layers measured by OCT
was similar to those measured by histology;
the muscular and connective-acinar structure
was visible until the working depth of
penetration into the tissue of the near-focus
probe (about 1 mm).
Smooth muscle structure appeared at OCT
scanning as hyper-reflective, longitudinal
strips within a context of hypo-reflective
tissue and were particularly recognizable at
the level of sphincter of Oddi. Veins, arteries
and secondary pancreatic ducts were also
identifiable by OCT, characterized by hypo-
or non-reflective, well-delimited areas (Figure
6).
The images acquired in this study provided
information on tissue architectural
morphology that could have only previously
be obtained with conventional biopsy. These
Figure 5. Magnification of an OCT image from the
normal main pancreatic duct wall, compared with
histology. From the surface of the duct, up to a depth of
1 mm, the following layers are recognizable: a) the
single layer of epithelial cells, approximately 0.04-0.08
mm thick, visible as a superficial, hypo-reflective band;
b) the connective-fibro-muscular layer surrounding the
epithelium, visible as a hyper-reflective layer
approximately 0.36-0.56 mm thick; c) the connective
and acinar structure close to the ductal wall epithelium,
visible as a hypo-reflective layer.
Figure 6. OCT image from the normal main pancreatic
duct wall visualized by linear array OCT: a portion of
an accessory duct is visible close to the pancreatic duct
(yellow arrow) as a non-reflective, well-delimited area,
with larger diameter than observed for blood vessels
(red arrow). From the lumen to the depth is it possible
to recognize: a, epithelium; b, fibro- connectival tissue;
c, acinar tissue.

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results suggest that OCT could become a
powerful imaging technology, enabling high-
resolution diagnostic images to be obtained
from the pancreato-biliary system during a
diagnostic ERCP procedure.
Although OCT images were obtained from
surgical specimens, the early evaluation
(within one hour from pancreatic resection) of
ductal OCT-imaged morphology was not
likely to have been influenced by artifacts
induced by autolytic tissue degradation
occurring in autopsy specimens, as reported in
previous studies [12, 17, 18]. The data
obtained ex vivo from this study can therefore
be considered useful for in vivo evaluation
too.
Pathological Pancreatico-Biliary Ductal
System
Pathological pancreatic ductal system has
been investigated by our group in humans in
two ex vivo studies [12, 18, 22] performed on
multiple surgical pancreatic specimens
obtained from patients with pancreatic head
adenocarcinoma.
In chronic inflammatory changes involving
the main pancreatic duct OCT still showed a
conserved three-layer architecture. However,
the inner, hypo-reflective layer appeared
slightly larger than normal (0.07-0.24 mm),
and the intermediate layer appeared more
hyper-reflective than in normal tissue; this is
probably because of the dense mononuclear
cell infiltrate. The back-scattered signal was
heterogeneous with marked hypo- or hyper-
reflectance in some sections.
The agreement between OCT and histology in
the definition of main pancreatic duct (MPD)
chronic inflammatory changes was poor
(27.7%).
The OCT pattern in presence of dysplasia of
the main pancreatic duct epithelium was
characterized by an inner layer markedly
thickened (0.49 mm), strongly hipo-reflective
and heterogeneous; this OCT finding is
probably due to the initial structural
disorganization (increased mitosis and altered
nucleus/cytoplasm ratio). The surface
between the inner and intermediate layers
appeared irregular. As in chronic
inflammatory tissue, dysplasia too gave
strong hyper-reflectance of the intermediate
layer, particularly in the part closest to the
inner layer. The outer layer did not differ
from other non-malignant conditions and
appeared homogeneously hypo-reflective.
However, in chronic pancreatitis and
dysplasia only in 62% of cases OCT and
histology were concordant. The K statistic
used to assess agreement between the two
procedures was equal to 0.059 for non
neoplastic MPD wall appearance.
Overall, normal wall structure and chronic
inflammatory or low-grade dysplastic changes
cannot be distinguished in 38% of the sections
because the architecture of the layers and
surface light reflection did not show a
characteristic OCT pattern.
In all sections with histologically proven
adenocarcinoma OCT showed a totally
subverted MPD wall architecture. The three
layers of the ductal wall and their linear,
regular surface, normally giving a
homogeneous back-scattered signal, were not
recognizable. The margins between the
connective-fibro-muscular layer and acinar
tissue were unidentifiable. The back-
scattering of the signal appeared strongly
heterogeneous, with minute, multiple, non-
reflective areas in the disorganized pancreatic
microstructure. In 100% of sections with
adenocarcinoma OCT and histology were
concordant. A totally subverted wall
architecture was also observed by OCT in
presence of neoplastic tissue within the
common bile duct [21].
Figure 7 shows magnified OCT images from
sections of main pancreatic duct with normal
tissue, chronic pancreatitis, low-grade
dysplasia, and adenocarcinoma.
Clinical Application of OCT in the
Pancreatico-Biliary Ductal System
Segmental strictures of the MPD may in fact
be difficult to investigate, particularly when
they are in the middle and tail of the gland. In
these cases a definite diagnosis can be
achieved only by cytology, either by
intraductal brushing or fine needle aspiration

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biopsy (FNAB). The diagnostic accuracy of
brush cytology tends to be low [23, 24, 25],
while FNAB, though it offers satisfactory
accuracy, is an invasive technique and usually
requires EUS as an additional procedure.
OCT could therefore be a useful, not
excessively invasive technique for
investigating in detail the MPD wall structure
in patients with segmental stricture, since the
probe can be inserted into the pancreatic duct
and segmental strictures through a standard
transparent ERCP catheter. Repeated frames
can be taken by the pull-back technique in a
few minutes, giving a large number of radial
and longitudinal images. The transparent
surface of the ERCP catheter did not
substantially affect the OCT diagnostic
capacity, compared with images obtained
keeping the probe outside the catheter.
The MPD has been investigated by OCT
during ERCP procedures by our group in a
series of 12 consecutive patients with
documented or suspected MPD stricture at a
previous CTscan or MRCP; all patients
underwent endoscopic ultrasonography before
ERCP [22]. OCT recognized a differentiated
three-layer architecture in all cases with
normal MPD or chronic pancreatitis, while in
all the neoplastic lesions the layer architecture
appeared
totally
subverted,
with
heterogeneous back-scattering of the signal.
OCT was able to distinguish a non-neoplastic
from a neoplastic MPD wall in all cases and
was superior to brush cytology; OCT gave
100% accuracy for detection of neoplastic
tissue compared with 66.7% for brush
cytology. The presence or absence of a
recognizable three-layer architecture within
the MPD wall, derived from the different
back-scattered signals from each layer, was
confirmed as a reliable OCT parameter for
distinguishing non-neoplastic from neoplastic
tissue, as already documented in previous
experimental studies. Despite the superiority
of the OCT to distinguish a non-neoplastic
from a neoplastic MPD stricture, the
technique appeared substantially unable to
discriminate between a normal MPD structure
and other benign lesions of the MPD. The
catheter sheet made it difficult to examine the
inner hypo-reflective layer of the MPD wall,
corresponding to the single layer of epithelial
cells of the MPD surface, particularly when
the MPD diameter was small. This occurred
particularly when OCT frames were obtained
inside the strictures, as the superficial
epithelial layer was compressed by the ERCP
catheter. However, the diagnostic capacity of
the technique was not substantially affected,
since the differential diagnosis between non-
neoplastic and neoplastic lesions was based
on identification of the layer architecture
(Figure 8).
OCT scanning performed within ERCP has
been also useful in identifying neoplastic
lesions at early stages involving the common
bile duct, missed by cytology and X-ray
imaging (Figure 9).
OCT imaging of the biliary ductal system has
been done in-vivo in two previous ERCP-
Figure 7. Magnified OCT images from sections with either normal (a.), tumor-associated chronic inflammatory (b.),
low-grade dysplasia (c.), and adenocarcinoma (d.) tissue.

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based studies [20, 21]. The first OCT
evaluation of the biliary ductal system was
done in four cases by Seitz et al. [21], who
demonstrated the feasibility of the technique
in patients with biliary pathology and
obtained interpretable images. The connective
tissue layer and the underlying retroperitoneal
tissue, with a reduced light back-scattering,
were clearly demonstrated, toghether with the
intramural glands, visualized as non-reflective
areas. The second in-vivo study by Poneros et
al. [20] showed the technique was feasible
during a convenctional ERCP in 5 patients
and recognized the normal OCT pattern,
compared to histology, of both intra- and
extra-hepatic biliary tree; OCT pattern of
epithelial and subepithelial structures,
including peribiliary glands, vascular and
hepatic structure were described. The Authors
also reported the OCT pattern of inflamed
biliary ducts and cholangiocarcinoma. The
normal bile duct was characterized by a
layered structure similar to that observed by
Authors in ex-vivo images. The superficial
cuboidal epithelium was observed as a thin,
hypo-reflective layer overlying an hiper-
reflective submucosa and a weakly-reflective
serosa. The inflammed bile duct was
described as heterogeneously-reflective
submucosal layer with dilated peribiliary
glands. OCT pattern of cholangiocarcinoma
was characterized by a villous morphology,
with an underlying fibrous stroma clearly
differentiable from the non-neoplastic tissue.
Future of the OCT in the Pancreatic-
Biliary Ductal System
Recently, a new linear-array OCT (LOCT)
has been proposed in Europe for experimental
use only, with an higher resolution rate (21-27
μm) than the radial one (ROCT), a same
frame acquisition and penetration depth. Up
to now, LOCT is allowed to be tested only in
animals and surgical specimens. In a pilot
study we evaluated the accuracy of LOCT,
compared with ROCT, in the recognition of
the normal CBD layers’ structure in a series
of surgical specimens from patients who had
undergone pancreatic head resection for
pancreatic cancer not involving the biliary
tree. Images obtained by LOCT and ROCT
were compared with the corresponding
histologic features (Figures 10). LOCT
images appeared superior to those obtained by
ROCT for the recognition of CBD wall
microstructure, particularly at the level of the
intermediate fibro-connectival submucosal
layer. The possibility to recognize in detail
the microstructure of the connective and
muscular layer could be useful in the
Figure 9. Adenocarcinoma of the common bile duct at
early stage detected by OCT during ERCP. Whithin a
normal tissue, OCT recognizes a delimited area
showing a subverted wall architecture, with loss of the
ductal layers and unidentifiable connective and
muscular layer. The three layers and their linear and
regular surface, normally giving an homogeneous
back-scattered signal, are not recognizable. This OCT
image has an heterogeneous back-scattered signal with
minute, multiple, non-reflective areas (necrotic areas)
in the disorganized CBD microstructure.

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