Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5528_Библиотеки_им_академика_М_И_Перельмана.pdf
110 S. Sherwani
(a) (b) (c)
(d) (e) (f)
(g) (h) (i)
Figure 24. Pseudomembranous colitis, CLE. (a) Endoscopic image showing cream-colored membrane overlying erythematous mucosa of pseudomembranous colitis. (b) Normal colon. (c, d) Mild
colitis with vascular proliferation and fluorescein leakage. (e) Cellular infiltrate and crypt distortion
in mild colitis. (f) Intraluminal and interstiti a l fluorescein leakage. (g, h) Severe changes of pseudomembranous colitis including fluorescein leakage, severe crypt distortion and destruction, and inflammatory infiltrate.
75
et al.
been demonstrated as acriflavine positive, fluorescein negative bright rods
embedded in the colonic mucosa.
75
Intestinal spirochetosis
Günther et al. described the interesting findings of colonic spirochetosis
using eCLE imaging.
promised patients presenting with watery diarrhea.
play an important role in targeting or detecting spirochetosis as it usually involves endoscopically normal appearing colon.
76
Spirochetosis may be suspected in immunocom-
76
CLE may therefore
76
With the aid of
Lower Gastrointestinal Tract 111
Figure 25. Collagenous colitis, CLE. Gray “shells" surrounding crypts in collagenous colitis.
78
acriflavine staining, spirochetes appear as a bright ring within the colonic
76
crypts.
Microscopic colitis
Collagenous colitis
Given the patchy nature of microscopic colitis, multiple studies explored
the utility of CLE as a tool for targeting biopsies.
tologic findings in collagenous colitis, CLE also demonstrates subepithelial thickening of the collagen bundles surrounding crypts and appearing as
77,78
dark gray “shells” (Figure 25).
Rarely, s uperficial crypt patterns maybe
slightly abnormal, however they are more likely to appear normal.
58,77, 78
Similar to the his-
77,78
Lymphocytic colitis
Lymphocytic colitis is characterized by patchy infiltrate of lymphocytes
often involving the superficial colonic epithelium. CLE imaging shows
massive infiltration of the mucosa (epithelium and lamina propria) with
112 S. Sherwani
et al.
Figure 26. Lymphocytic colitis, CLE. Lymphocytic colitis seen as aggregates of dark cells expanding
the lamina propria.
79
small dark lymphocytes forming sheets between the crypts.58Damage to
the surface epithelium may be seen, howeversuperficially cryptarchitecture
and distribution are often preserved (Figure 26).
58,79
Graft-versus-host disease (GVHD)
CLE examination of the small intestine may allow for rapid evaluation of
transplanted intestine.
in concert with transplantation. Villous destruction is the hallmark feature
of acute rejection.
villous epithelial border, luminal fluorescein leakage, enterocyte shedding,
and reduction in goblet cell density.
Coron et al. characterized the features of graft versus host disease
using pCLE imaging.
if either abnormal microvasculature or mild lamina fluorescein leakage
80
Acute rejection can be monitored endoscopically
80
Features of villous destruction include an irregular
80
81
In their classification, GVHD was defined as mild
Lower Gastrointestinal Tract 113
(a) (b) (c)
(d) (e) (f)
Figure 27. Graft-versus-host disease, CLE. Grades of GVHD: (a) Normal colon. (b) Abnormal
microvascular network. (c) Mild fluorescein leakage. (d). Distorted crypts. (e) Severe microvascular
distortion. (f) Severe architectural destruction.
81
was seen.81If both features were identified or if mild architecturaldistortion
is present, GVHD is graded as moderate.
81
GVHD is graded as severe if
either severelamina fluorescein leakage or severedestruction of crypts were
identified (Figure 27).
81
Neoplasia
Microscopic in vivo imaging in the lower gastrointestinal tract does not
specifically aim for the detection of lesions (this usually requires a wide
field technique) but for on-site high-resolution characterization of lesions
detected by other means.
needs: First, visualization of ultrastructural details that are normally not
revealed with high-definition colonoscopy as a method not only to predict
but to actually visualize histology, second, for risk stratification and on-site
developmentof a therapeuticstrategy,and third, in conjunction with molecular imaging in a translational approach (at least at present). Computeraided diagnosis aims at supporting the non-expert/non-histopathologist
with analysis of microscopic features.
82
On-site characterization serves several clinical
114 S. Sherwani
et al.
Morphology
Starting from magnification or zoom endoscopy, it has been the ultimate
goal of endoscopists to revealmicroscopic structures in a resolution close to
conventional histology. Importantly, the orientation of the optical sections
of most in vivo microscopy devices is parallel to the tissue surface. This is in
contrast to conventional ex vivo histology which usually provides sections
◦
at a 90
topical application of a fluorescent contrast agent. For endocytoscopy (EC),
topical intravital stains are preferred. In endoscope-based CLE (eCLE),
sections can be obtained in differentdepths by a z-axis actuatoron the endoscope. In probe-based CLE (pCLE), changing pressure onto the mucosa and
different probe types allows different imaging plane depths. Whereas systemic staining allows exploitation along the full range of the z-axis, topical
staining yields superficial optical sections.
orectal lesions based on tissue structure (at superficial sections) and vessel architecture (at deeper sections) after injection of fluorescein. With
this classification,normal, regenerative (inflammatoryor hyperplastic),and
neoplastic changes could bedifferentiated with99% accuracy in earlyeCLE
trials.
their tubular or villous architecture. Whereas normal mucosa shows a regular array of hexagonal crypts, finger-like protrusions are seen in adenomas.
They are lined with a regular array of a single layer of high prismatic
cells, including goblet cells. In deeper sections, the vessels of the Lamina
propria become visible. For pCLE, a similar comprehensive classification
system has been published.
so some of the classical features to determine the grade of dysplasia (e.g.
nuclear-to-cytoplasmic ratio) can only indirectly be determined.Acriflavine
stains nuclei. It is applied topically and sprayed onto the mucosa, yielding
a superficial cellular resolution (Figure 28(d)). Acriflavine has been used
to differentiate high-grade from low-grade neoplasias in vivo.
no adverse events have been reported (and acriflavine and similar chemical compounds are freely available, e.g. for cleaning of fish tanks), positive staining of nuclei may indicate nuclear interaction. Therefore, negative
angle to the tissue surface.
Confocal laser endomicroscopy (CLE) usually requires systemic or
eCLE has been key to the development of a classification of col-
83,84
As exemplified in Figure 28, adenomas can be visualized with
85
Importantly, fluorescein does not stain nuclei,
86
Although
Lower Gastrointestinal Tract 115
(a) (b)
(c) (d)
Figure 28. Colorectal adenoma, CLE. (a) In colorectal adenoma, a single-layered epithelial layer
is seen, containing regular high prismatic cells with interspersed goblet cells. Crypts are elongated
(tubular adenoma). (b) In deeper sections, resolution of the epithelium is less accurate, but the lamina
propria is more clearly defined, containing bright fluorescein-filled vessels with black dots, indicating
unstained erythrocytes. (c) With higher grades of dysplasia, superficial optical sections identify slightly
more irregular cells — note that nuclei are not visible, but darker tissue is suspicious for neoplasia.
The brightly stained lumen identifies barrier dysfunction, i.e. micros copic bleeding/fluorescein leakage
from the lesion. (d) With acriflavine, superficial sections reveal the structure, including nuclei, of a
tubular adenoma. Within the lamina propria, dark vessel lumens are visible in between the elongated
crypts, and the endothelial cells can be identified. eCLE, 1a, b, c: fluorescein iv.,1d: topical acriflavine;
edge length 475µm. Images courtesy of Dr. Martin Götz.
nuclear stains havebeen tested. Cresyl violet was applied topically in a pilot
23
trial.
Sensitivity and specificity for the differentiation of non-neoplastic
from neoplastic lesions were 98% and 100%, respectively, however with
small numbers of patients. Whereas fluorescein and acriflavine yield bright
images at 488 nm (the wavelength used by CLE), cresyl violet excitation
116 S. Sherwani
et al.
and emission spectra peak at 590 and 630 nm. Therefore, images are less
contrasted, and laser power has to be maximized. In colorectal carcinomas, the tissue architecture is progressively lost (Figure 29). Cellular size
is irregular, and in deeper optical sections, tumor vessels become visible. In
addition, two distinct features are seen after fluorescein injection: Leakage
of contrast from tumor vessels into the tissue and into the lumen becomes
more prominent and malignant cells are darker than non-malignant cells.
The latter is due to pH-dependent fluorescence intensity of fluorescein and
more acidic, lower pH in tumor cells.
Endocytoscopy is fundamentally different from CLE from a technical point of view in that it constitutes an ultrahigh magnification based on
light microscopy. Only few groups had access to these prototype endoscopes, limiting the widespread use in clinical routine despite their quasihistological resolution and staining options. For the colorectum, different
staining methods have been tested. In a prospective small series, crystal
violet 0.05% was tested vs methylene blue 1% vs a combination of both.
The authors concluded that double staining is optimal for both nuclear and
crypt analysis.
33
In a prospective trial, on-site analysis by endocytoscopy
was evaluated vs. routine histopathology for 102 vs 101 lesions, respectively. Endocytoscopy was non-inferior to histopathology, with the advantage of providing immediate microscopic information.
87
Endocytoscopy
was also able to elicit features for differentiation of serrated lesions: Larger,
oval lumina were characteristic of sessile serrated adenomas (SSA, corresponding to a type II-o pit pattern), wherea s star-shaped lumina predicted
88,89
hyperplastic lesions (corresponding to conventional type II).
Optical
biopsy in SSA was also possible by pCLE, among other criteria visual-
90
izing an increased number of goblet cells.
In ulcerative colitis, a study
has combined chromoendoscopy for the detection of lesions with eCLE
for immediate characterization.
69
The presence of neoplastic lesions
could be predicted with 97.8% accuracy based on the above-mentioned
classification.
Risk stratification
The use of in vivo microscopy devices for identifying high-risk lesions
mainly aims at elaborating superficial signs predicting deep submucosal
Lower Gastrointestinal Tract 117
(a) (b)
(c) (d)
Figure 29. Colorectal cancer, CLE. (a) In colorectal cancer, the tissue microarchitecture is progressively lost: A residual crypt-like structure can be seen on the left side of the image, with leakage from
tumor vessels in the upper part. Black crowded cells are forming pseudo-cryptal buds that are visualized in the right lower corner. (b) With cresyl violet, malignant cells (upper part of the panel) within an
adenoma (lower part) are seen. Note that the nuclei are discernible. (c) In colitis-associated neoplasia, neovascularization is even more prominent, correlating to the fact that inflammation per se is a
trigger for enhanced vascularity, as is the inflammation-dysplasia sequence. (d) In colitis-associated
neoplasia of the distal rectum, the neoplastic changes (left side, with residual elongated crypts
and interspersed dark goblet cells) undermine the squamous epithelium (right side of the panel) at
the anorectal verge. Note the darker cells (less fluorescence in malignant cells) undermining the
squamous epithelium at 5 o’clock and the fluorescein leakage. eCLE, 1a–c: intravenous fluorescein,
1d: topical cresyl violet; edge length 475µm. Images courtesy of Dr. Martin Götz.
118 S. Sherwani
et al.
invasion, i.e. colorectal cancer of ≥T1a. This should prevent unnecessary
resection attempts and the inadvertent effects of complications (e.g. perforation secondary to deep invasion).
With CLE, studies have tried to quantify angiogenesis to predict colorectal cancer. Ex vivo, both vascular diameter and density were significantly increased in colorectal cancer.
91
With pCLE, 83 in vivo captured
videos of 51 lesions from 31 patients have been analyzed. Nests of dark,
irregular cells with irregular architecture and mucin depletion were found
92
in submucosal carcinoma infiltration.
In order to predict the depth of invasion, Kudo et al. have evaluated endocytoscopy criteria to determine mas-
sive submucosal invasion. Unclear glandular lumina, significantly enlarged
nuclei, and loss of single-layered stratification were predictive of deep
93
submucosal invasion.
Endocytoscopy has recently been combined with
narrow-band imaging (NBI) in a prototype endoscope for detailed vascular analysis.
94
Vasodilatation, change of vessel caliber, loss of the micronetwork pattern, and beading of vessels were associated with submucosal
deep invasion (Figure 30). The overall accuracy of NBI-augmentedendocytoscopy was 97%, although interobserver agreement for experienced investigators was somewhat lower and ranged between kappa = 0.44 and 0.67
for the above-mentioned criteria.
Molecular imaging
Molecular microscopic imaging refers to the characterization of lesions
based on their molecular fingerprint.
from a phage library, colonic adenomas were specifically visualized by
pCLE after topical application in patients.
cancer and in ex vivo specimens, epidermal growth factor receptor (EGFR,
Figure 31)
98
and vascular endothelial growth factor (VEGF)99have been
used to characterize neoplastic lesions and to quantitate target expression
by eCLE. This has been transferred to in vivo molecular imaging in 37
patients by topical application of labeled antibodies. An EGFR-specific
signal was documented in 18/19 colorectal cancer lesions and in 12/18 adenomas, whereas no or only weak fluorescence signal was noted in normal
mucosa.
100
In animal models, such quantification based on specific fluo-
rescence was predictive of response to targeted chemotherapy.
95,96
With an oligopeptide derived
97
In mouse models of colorectal
101
Tumor
Lower Gastrointestinal Tract 119
Figure 30. Endocytoscopic criteria of deep submucosal invasion.
Figure 31. Molecular imaging, CLE with immunohistochemistry.98(B) Tissue sample incubated with
EGFR and visualizedvia immunofluorescence. (D) Routine immunohistochemistry for EGFR. (A) and
(C) are omitted.
94
microvessels were specifically visualized in tissue samples by eCLE with
CD105 immunostaining,
orescently visualized in LGR5-EGFR transgenic mice.
102
and intestinal cancer stem cells have been flu-
103
Computer-aided diagnosis (CAD)
Most gastroenterologists are not explicitly trained to interpret mucosal
pathology from microscopic images, and histopathologists are usually not
available for on-site microscopic diagnosis. Therefore, CAD algorithms