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© 2024 World Scientific Publishing Company
https://doi.org/10.1142/9789813206984_0014
Hepatobiliary System Chapter
14
Erin Rubin∗, James Park Dewar∗,
Daniel Schmolze
Introduction
†
, and Wei Zheng
‡
Second to the skin, the liver is the largest and heaviest organ in the human
body. There are many functions of the human liver. These are not limited to,
but include, the metabolism of food, alcohol, andmedications, the storageof
nutrients, and cleaning waste. Approximately 80% of the blood delivered to
the liveris through the portal vein, filled with blood high in nutrients;20% is
provided via the hepatic artery,rich in oxygenated blood. The liver produces
bile, albumin,and most of the clotting factors.There are many cell types that
comprise this organ, with the majority being the parenchymal epithelial cell
hepatocytes. There are biliary epithelial cells and non-parenchymal cells
∗
Department of Pathology and Laboratory Medicine, University of Texas Health Science
Center at Houston, Houston, TX, USA.
†
Department of Pathology, City of Hope, Duarte, CA, USA.
‡
Department of Pathology and Laboratory Medicine, Emory School of Medicine, Atlanta,
GA, USA.
281
282 E. Rubin
et al.
including hepatic stellate cells or Ito cells. The liver sinusoids are lined
with sinusoidal endothelial cells and phagocytic Kupffer cells. Each of
these cell types has the potential to become neoplastic. The most common
benign tumor of the liver is a cavernous hemangioma and the most common
primary malignant tumor of the liver is hepatocellular carcinoma. Many of
the diseases of the liver are caused by alcohol use, metabolic syndrome, and
hepatitis viral infections: most commonly hepatitis B virus or hepatitis C
virus. Liver disease is a major cause of mortality and morbidity worldwide.
Accurate diagnosis is essential for proper treatment.
The liver is found in the right upper abdomen, below the diaphragm
and under the ribs. Imaging of solid organs remains a challenge due to
accessibility. Recent advances in biological imaging devices provide opportunities in the field of transplantation for pre-transplant donor evaluation
of organ viability and intraoperative ly during laparoscopic or open surgery.
Whole-body, non-invasive, radiologic imaging techniques have rapidly
advanced to include Positron emission tomography (PET), MRI, and CT.
The resolution, however, of these modalities is limited. Histopathologic
analysis is still a mainstay in the diagnostic workup of liverdiseases, including neoplastic and non-neoplastic diseases. The gold standard of microscopic imaging remains the formalin-fixed, paraffin-embedded (FFPE),
4 µm tissue section stained with hematoxylin and eosin (H&E). For the
liver, the ideal specimens are obtained using 15- or 18-gauge core needle
biopsies. There are several drawbacks to performing a core needle biopsy
of the liver. The FFPE technique is invasive and there is a time delay. The
image is static and represents only one time point. Only after 4–12 hours
can early coagulative necrosis be seen by H&E analysis.
The most rapid, microscopic, diagnostic technique used in clinical
settings is the cryosection (or frozen section). This technique is used for
intraoperative assessment of margin status during tumor resections, pretransplant donor evaluation, and other time-sensitive evaluations. Cryosections aremore diagnostically challenging,with more falsenegative and false
positiveresults than FFPE sections. In vivo imaging modalities are superior
to both FFPE and cryosections in that they are real-time non-invasive and
result in little tissue injury. More than cryosection, in vivo imaging has the
potential to provide insight into functionality, viability, necrosis, and adequacy of flow. There is less risk of sampling error as it is easier to image a
Hepatobiliary System 283
larger surface area rather than the limitations of a single sample. There are
no freezing or cutting artifacts or tissue loss, as seen in the intraoperative
construction of the H&E stained cryosection.
Non-invasive, high-resolution imaging of the liver is an attractive
prospect for several reasons. A core needle biopsy carries an increased risk
of bleeding, particularly in patients with coagulation abnormalities. Most
of the clotting factors are made by the liver. Fear of bleeding may prompt
smaller biopsies, which may be non-diagnostic or inaccurate.
1, 2
Second,
random percutaneous liver biopsy can lead to sampling error, even for dis-
3,4
eases which involve the entire liver, such as hepatitis.
Mini-laparoscopy
can help target biopsies and has been shown to improve diagnostic accu-
5, 6
racy,
but an invasive biopsy is still ultimately required.
Recent advances in optical imaging technologies have become a significant source of innovation in histopathologic diagnosis. This exciting
new technology provides pathologists and clinicians with a potentially new
way to visualize and generate diagnoses in patients, real time, both in vivo
and ex vivo. A growingarray of optical imaging techniques have been investigated as alternatives or as adjunct approaches to diagnosis. This powerful
technique will potentially allow for the clinical assessment of hepatic diseases at the microscopic level, both real time and in vivo.Atthistime,
the techniques include optical coherence tomography, conventional confocal microscopy, and confocal endomicroscopy. These promising imaging
modalities go beyond hematoxylin and e osin (H&E) histopathologic evaluation by offering an instant, minimally inva sive, or non-invasive diagnosis
in tissues (the so-called optical biopsy). These can also provide dynamic
monitoring and screening of pathologic tissue to guide targeted tissue sampling and to improve diagnostic accuracy.
As in most solid organs, in vivo microscopy (IVM) of the liver is
at a relatively early stage of development. Unlike the skin, gastrointestinal and genitourinary tract, most solid organs are not readily accessible.
Other barriers to IVM include limited depth of penetration, photobleaching, phototoxicity, image quality, the large size of imaging devices, and
how to maintain sterility. The limitation of the depth of penetration has constrained in vivo imaging to a few hundred microns below the liver surface.
In non-neoplastic diseases with global liver involvement, such as hepatitis,
this limitation is less important. In vivo imaging has been successfully
284 E. Rubin
et al.
applied in such diseases, with promising early results. These studies have
used confocal laser endomicroscopy (CLE) by adapting the devices now
widely availablefor use in the tubular gastrointestinaltract. Neoplastic liver
applications have so far been limited to either animal models or ex vivo
experiments. As in vivo imaging devices continue to become smaller and
more flexible, this field will advance faster. New avenues for imaging small
branches of the biliary tree or hepatic vasculature and hepatic neoplasms
will become feasible. This chapter will focus on the major advances of
in vivo and ex vivo studies in humans and animals, as well as preclinical
evidence of diagnostic capacity and potential clinical applications of these
imaging technologies. This chapter will assess the strengths and limitations
of each approach, specific to hepatobiliary pathology, with an eye toward
possible future developments.
Optical Coherence Tomography (OCT)
As fully discussed in other chapters, OCT is an interference-based optical
imaging modality, which fills a resolution gap between ultrasound imaging and confocal endoscopic imaging. The image resolution of standard
OCT is similar to a 4x objective (N.A. 0.1) of a light microscope (approximately 2–10 µm). Although this resolution is not sufficient for routine
histopathologic diagnoses, OCT is advantageous in that it provides a good
penetration depth of up to 2–3 mm. OCT allows assessment of different
layers of tissue components with a relatively high resolution (10 to 100
times finer than standard clinical ultrasound). OCT has been applied to
image human as well as animal liver tissue ex vivo.
OCT has been used to image biopsy specimens and has been found to be
able to distinguish normal, early cancer, and cancerous liver tissues. The
histological architecture and bland cytology of normal liver can be readily visualized by en face tomographic imaging, including central veins,
hepatocyte cords, sinusoidal spaces, and the portal triad (portal vein, hepatic artery, and bile duct). OCT imaging of a wide variety of histological
subtypes of hepatocellular carcinoma was able to identify malignant cytological features, particularly nuclear atypia (enlarged convoluted nuclei)
and thickened fibrous bands.
8
Ex vivo OCT imaging of human normal liver
7
In the research setting,
Hepatobiliary System 285
tissue revealed intrahepatic bile duct, including biliary epithelium, subepithelial lamina propria, and fibroadipose connective tissue.The surrounding
hepatic parenchyma had a homogeneous contrast and was seen underlying
the connective tissue.
9
In contrast, visualizing intrahepatic cholangiocarcinoma using OCT exhibited a unique feature of the portal vein showing
a blurred pattern in the lateral direction and an aggregated distribution in
the axial direction.
8
A recent human pilot study extended OCT’s applicationtothein vivo level. The study performed endoscopic OCT imaging
of the intrahepatic biliary tree and showed the feasibility of OCT in distinguishing non-neoplastic from neoplastic lesions in vivo. Biliary ductal
epithelium and subepithelial structures, including peribiliary glands, vasculature, and hepatic parenchyma were observed. In two c ases of papillary
cholangiocarcinoma, OCT was able to identify the characteristic papillary epithelial architecture and quantify epithelial thickness without OCTrelated complications.This preliminary study is highly meaningful because
the diagnosis of bile duct lesions obtained by traditional ERCP biopsy is
often non-diagnostic. The sensitivityof intraductal brush cytology is highly
variable. In addition, endoscopic OCT can provide broad-field, subsurface,
and near-microscopic imaging of hepatobiliary tract in real time during the
endoscopy procedure. These results suggest that OCT has the capacity and
potential to reduce sampling errors and improve diagnostic sensitivity by
identifying suspicious lesions and guiding targeted biopsies and surgical
interventions. As the usage expands and more validation studies are conducted with OCT in evaluating hepatobiliary lesions, we expect that OCT
will play a more significant impact on the clinical diagnosis and management of liver diseases.
Conventional Confocal Microscopy and Confocal Endomicroscopy
Reflectance confocal microscopy utilizes a near-infrared laser beam to
illuminate tissue and detects the natural differences of tissue refractivity,
without the application of exogenous contrast agents. Any structure with
high refractivity is displayed as white on a computer monitor, whereas any
substance with no refractivity is visualized as black. Reflectance confocal
286 E. Rubin
et al.
microscopy has been utilized to assess liver parenchyma both in vivo and
ex vivo. In vivo reflectance confocal images from a series of normal rat
livers readily revealed essential architectural elements such as hepatic cell
plates, sinusoids, venules, and portal vein spaces. Canaliculi could also be
identified. However, defining the limits of the basic parenchymal unit was
not successful because reflectance confocal does not allow recognition of
differences in refractivity between hepatocytes of different acinar zones.
Due to low resolution, the evaluation of hepatocyte intracellular detail was
unsatisfactory.
10
Both fibrosis and steatosis were able to be evaluated using
ex vivo reflectance confocal microscopy in both diseased and non-diseased
states froma series of 12 human liver biopsy samples.Steatosis wasdepicted
as well-demarcated, non-refractive globules of various sizes, ranging in
diameter from 7.2 to 29 µm. Portal tract fibrosis was observed as the coarse
bundles or networks of highly refractive materials in the portal space.
11
Common types of primary and secondary hepatic tumors were also satisfactorily evaluated with reflectance confocal microscopy, showing loss of
hepatic architecture and newly formed structures, such as large vascular
spaces, reactive fibroblasts, calcification, and inflammatory infiltrates.
Fluorescence confocal microscopy uses laser illumination to excite
exogenously-induced fluorescent probes and collects excited signals from
the tissue, allowing specific structure labeling and imaging. It is most commonly implemented form of confocal microscopy. Obstacles of in vivo
imaging include photobleaching and phototoxicity, which is inherent to
any technique that uses fluorescence. The imaging techniques, although
higher in resolution than radiologic imaging, are not able to view deeply
into the tissue. So, for example, if the liver capsule is fibrotic, assessment
of the tissue deep to the capsule is impaired. Imaging of a cross-section,
wedge, or needle biopsy is possible, however.
Conventional fluorescent confocal microscopes are bulky instruments
normally used in research laboratory settings. This imaging technique has
been used to study almost all types of hepatobiliary diseases in human and
in animal cell lines for decades by introducing various fluorescent labeling
molecules. However,it was only a recent developmentto apply conventional
confocal microscopy for in vivo imaging of the hepatobiliary system. By
using fluorescent probes to label multiple cells and molecules simultaneously in vivo on animal models, this new methodology allows researchers
Hepatobiliary System 287
to observe different hepatic parenchymal cells in their native environment.
Hepatocytes, endothelial cells, and leukocytes were able to be visualized
at the cellular or even subcellular level. With this technique, researchers
are able to track temporospatial changes in morphology, metabolism, and
function of hepatobiliary cells under various physiological or pathological
conditions.
12
Recent advances in instrument miniaturization have led to the development of fluorescein-aided, miniprobe-based confocal laser endomicroscope (CLE). This flexible, optical fiber can be placed through the working
channel of standard endoscopes or laparoscopes. Moreover,in recent years,
confocal laser-based imaging devices have been developed that are small
enough to fit within a biopsy needle, enabling so-called needle-based confocal laser endomicroscopy (nCLE). This exciting advancement for in vivo
microscopy of solid organs has great potential applications, such as more
accurate targeting of biopsies. Despite a fixed imaging depth and relatively
low resolution of CLE, faster image acquisition and real-time in vivo histology examination during ongoing endoscopy or surgery have prompted
the exploration of the application of this technique in hepatobiliary imaging and diagnosis. Animal studies do not have the limitations and safety
issues of human trials, so many fluorescent contrast agents were first investigated on animals and were later rapidly transferred into human applica-
13
tions.
For example, one initial animal trial in normal mouse liver revealed
that CLE, with intravenous application of fluorescent contrast acriflavine,
clearly identifiedthe lobular patternof the hepatic cells and surfaceendothelial cells. Subnuclear details of liver cells were also visible. The study
also demonstrated that CLE images were comparable with corresponding
14
10µm-thick hematoxylin–eosin-stained tissue images.
Another early
in vivo animal study used CLE to image dense mononuclear inflammatory
infiltration in IL-12-induced hepatitis model and to visualize bile ductular
proliferation and bile canaliculi dilation in a common bile duct ligation
15
model.
The CLE histologic images correlated well with conventional
histology. A subsequent animal trial investigated the role of CLE in the
evaluation of cirrhosis. After intravenous injection of fluorescein and using
a probe of 50 or 100 µm working distances, CLE was able to visualizecords
of hepatocytes radiating toward central venules in normal livers and distorted hepatic architecture was observed in cirrhotic rats. Moreover, CLE
288 E. Rubin
(a) (b) (c)
et al.
(a)
(a)
Figure 1. In vivo nCLE imaging of normal and cirrhotic rat livers. (Top row) Regular cords of
hepatocytes are seen radiating toward a central venule in healthy livers. (a) = 0 µm probe,
(b) = 50µm probe, (c) = 100µm probe. (Middle row) Images from cirrhotic livers, acquired from
within the hepatic parenchyma, show irregular clumps of cells separated by bright bands. (Bottom
row) a distinctive reticular pattern, seen in cirrhotic livers but only at the surface with the 0µm probe.
Adapted from Ref. [16].
(b) (c)
with a probe of 0 µm working distance identified a unique reticular pattern
on the liver surface that was only found in cirrhosis (Figure 1). This suggested that CLE could provide sufficient detail to distinguish normal from
cirrhotic livers.
16
Another animal study using dual wavelength CLE in vivo imaged fluorescein at 488 nm and indocyanine green at 660 nm in a porcine model
of laparoscopic microwave liver ablation. The study showed that dual
wavelength CLE permitted the distinction of in vivo of thermally ablated
Hepatobiliary System 289
liverfrom healthy liverduring laparoscopic surgerybased on loss of fluorescence intensity. CLE imaging of fluorescein provided good visualization
of the hepatic microvasculature; whereas, CLE imaging of indocyanine
green clearly displayed the hepatic sinusoid architecture and interlobular
septations.
17
A limited number of experiments have investigated the potential of
in vivo microscopy to obtain temporal information, whether in the liver or
in any other organ system. These capabilities of IVM tend not to translate
neatly into pre-existing diagnostic frameworks but offer perhaps the most
potential for novel insights into disease processes.
One such study has been performed in the liver, using a mouse model
to examine both the spatial and temporal patterns of hepatocyte apoptosis.
21
Three different fluorophores were used: acriflavine (to provide hepatocyte
contrast), FITC-labeleddextran (predominantly used to investigatechanges
in vascular permeability), and fluorescently labeled poly-caspase inhibitor
(FLIVO). Following Jo2-induced apoptosis, images were consecutively
acquired from the same region of the liver surface at 5–10 minute intervals
for up to 4 hours. Detailed sequential morphologic changes of apoptosis
were observed, including the initial formation of cell membrane vesicles,
cell swelling, and eventual nuclear blebbing and pyknosis (Figure 2).
Representative Human Confocal Laser Endomicroscopic Studies
The first report of the application of CLE for in vivo imaging of human
liver was in 25 patients with suspected liver disease.
intravenously administrated as a contrast agent during laparoscopy. Basic
features of liver microarchitecture could be clearly visualized. In fatty liver
disease, liver steatosis was reliably identified and graded by a black appearance due to lack of fluorescent signal. Macrovesicular or microvesicular fat
inclusions could be distinguished from single-cell necrosis.
In vivo evaluation of fibrosis and cirrhosis was achieved by the identification of connective tissue strands, of capsular and pericellular fibrosis,
and of a micronodular liver surface by virtue of fluorescent labeling of
elastic and collagenous structures (Figure 3).
18
Fluorescein was