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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5528_Библиотеки_им_академика_М_И_Перельмана.pdf
90 S. Sherwani
et al.
Figure 4. Schematic of confocal laser endomicroscopy.
7
the pinhole will reach the detection system. This detected, reflected light
represents the desired focal plane, which the user of the system can manipulate without manipulating the tissue, hence its theoreticalutility to an in vivo
application (more to follow).
3,6,7
The detection system and on-board soft-
ware digitize this detected, reflected light into cross-sectional microscopic
8, 9
images.
of the device.
In vivo devices rely on suction to steady tissue against the surface
1
Historically, there have been two primary CLE techniques for the
GI tract: endoscope-based (eCLE) and probe-based (pCLE) (Figure 5).
10
eCLE, which is generally no longer in use, afforded endoscopists the ability
to select focal planes up to a depth of 250 µm (pCLE fixed at manufacturerdependent depth) as well as broader fields of view and better resolution
than pCLE.
11–14
However, eCLE devices suffered from lower frame rates
(i.e. choppier video) than pCLE and, owing to contractual arrangements,
required a dedicated location in already crowded endoscopes, giving them
a longer working length. pCLE — on the other hand — traverses an endoscope’s working channel and can thus be used with any make of endoscope
without affectingworking length. The combination of lower frame rates and
longer working lengths begot longer procedure times for eCLE ultimately
Lower Gastrointestinal Tract 91
(a)
(b) (c)
(d)
Figure 5. Probe- Versus Endoscope-Based Confocal Laser Endomicroscopy.10(a) Probe-based
confocal laser endomicroscope passes through working channel of endoscope and has fixed imaging
depth. (b) Endoscope-based confocal laser endomicroscope embedded in standard endoscope and
has adjustable imaging depth. (c ) Blue laser light. (d) Demonstration of focal plane differences
between A and B.
leading to poor adoption.
15,16
Also, the superior flexibility of pCLE, when
coupled with double-balloon enteroscopy, which uses inflation and deflation to navigate farther than traditional endoscopes, allows for better visualization of the mucosa of the small intestine through the mouth or colon.
17
All GI CLE techniques leverage intravenous 10% fluorescein as a contrast agent. Unbound fluorescein traverses capillaries staining extracellular
matrix, lamina propria, and surface epithelium.
8
Fluorescein dosing is variable in the literature with some studies employinga volume-based approach
to optimizing peak signal-to-noise ratios and safety (5 mL superior to
smaller doses),
(0.5–2.0 mg/kg).
18,19
while others have employed weight-based approaches
20,21
Figure 6 demonstrates one example of a weight-based
approach. The primary additional consideration is when to image postcontrast administration, which also varies between studies, but is generally
fewer than 10 minutes.
18,22
Fluorescein is rapidly metabolized by the liver;
its metabolites are mainly eliminated by the kidneys within 48–72 hours.
Side effects in GI CLE are minimal. One study of 2272 patients resulted
92 S. Sherwani
(a) (b)
(c) (d)
et al.
Figure 6. Fluorescein, CLE. (a) 0.01 mL/kg. (b) 0.02 mL/kg. (c) 0.05 mL/kg. (d) 0.10 mL/kg.
21
in 12 who experienced transient hypotension, 9 who had nausea, and no
serious adverse events.
offers indirect (negative) nuclear staining.
nuclei but is carcinogenic to humans.
18
Topical cresyl violet stains the cytoplasmand thus
24
23
Topical acriflavine can stain
CLE of normal lower gastrointestinal tract
Figures 7 and 8 demonstrate normal findings in the small intestine, including correlation with histology.
the aforementioned microanatomy save for muted nuclear features. As a
reminder, traditional CLE techniques acquire images in a plane parallel
to the surface of the intestine, whereas traditional histological techniques
demonstrate tissue in a plane perpendicular to the intestinal surface.
22,25
CLE findings generally recapitulate
(a)
(b)
Lower Gastrointestinal Tract 93
Figure 7. Duodenum, CLE. Normal duodenal villi post-fluorescein. (a) Enterocytes (arrows) and
goblet cells (arrowheads). (b) Vasculature of lamina propria.
25
94 S. Sherwani
(a)(
)
Figure 8. Terminal ileum, CLE, and histology. Normal villus of terminal ileum. (a) Confocal laser
endomicroscopy (CLE). (b) Histology. Enterocytes, goblet cells (arrows), and stroma observed with
equal resolution save for generally inconspicuous nuclei by CLE.
et al.
b
22
Figures 9–12 demonstrate normal findings in the colon, including
correlation with histology.
22,26, 27
The s ame axioms discussed in the above
paragraph on the small intestine hold for the colon. Additionally, CLE
imaging of the colon adheres to the following naming convention: surface
layer, intermediate layer (from just below the surface to a depth of 100 µm),
and deep layer (from the end of the intermediate layer to a depth of 250 µm).
By this convention, the surface refers to that which contacts the CLE probe,
which doesn’t always correspond to the surface of the epithelium (e.g.
within lumens where probes cannot reach).
22
Limitations of CLE
This section serves as a quick reference and includes previously discussed
concepts as well as several additional ones. To reiterate, CLE does not
generally visualize nuclei or subcellular details. Likewise, it does not generally resolve structures below the intestinal mucosa, thus rendering the
identification of tumor inva sion a challenge. Obviously, if one endeavors
to forgo conventional histology in light of CLE findings, one will not have
Lower Gastrointestinal Tract 95
Figure 9. Colon, surface layer ,CLE. Post-fluorescein. Each crypt has a slit-likelumen and is lined by
goblet cells (black dots represent mucin) and enterocytes. Between the crypts is a hexagonal network
of capillaries. The lamina propria is otherwise inapparent.
26
(a) (b)
Figure 10. Colon, intermediate layer, CLE, and histology. Post-fluorescein. Each crypt (black circle
represents one crypt) now has a round lumen, is lined by goblet cells with more elongated mucin
vacuoles (dark cylinders identified by white arrows), and contains fewer enterocytes relative to
Figure 9. The same hexagonal capillary network can be seen along with occasional foci of lamina
propria matrix (red box).
22
tissue for ancillary studies (e.g. immunohistochemistry or sequencing). As
with all endoscopic techniques, sufficient bowel preparation is essential
(Figure 13).
6
Likewise, adequate training and experience are needed to
ensure the CLE probe maintains contact with tissue surfaces and thus avoid
96 S. Sherwani
(a) (b)
et al.
Figure 11. Colon, deep layer, CLE, and histology. Post-fluorescein. Each crypt (black circle represents one crypt) is smaller in diameter and has a pinpoint lumen. The hexagonal capillary network
is less apparent owing to a relative increas e in the distribution of fluorescein in the lamina propria
versus the cytoplasm of the epithelium. The smaller crypts beget a relative expansion of the l a mina
propria (red box represents one focus).
22
Figure 12. Colon, deep layer, red blood cells in capillar i es, CLE. Post-fluorescein. Arrows point to
red blood cells (black dots) in capillary lumens.
27
Lower Gastrointestinal Tract 97
Figure 13. Content between probe and tissue, CLE. Insufficient contact caused by mucus, fecal
content, or air bubbles results in dark spots.
6
Figure 14. Motion artifact, CLE. Insufficient contact owing to patient, physiologic, or operator
movement.
6
98 S. Sherwani
et al.
Figure 15. Small intestine, OCT. Insufficient contact for uniform visualization of mucosa. The marks
on the vertical and horizontal axis are 1 mm apart. MM: Muscularis mucosa.
28
motion artifact (Figure 14).6Lastly, although criteriafor assessing abnormal
states exist, standards by which to assess operator competence have yet to
be established — an important point given the absence of such training
from traditional post-graduate training.
Optical Coherence Tomography
OCT, although central to the in vivo microscopic evaluation of other organ
systems and even the upper gastrointestinal tract, has not gained significant
footing in lower gastrointestinal tract applications owing to its inability
to easily and uniformly visualize the entire circumference of the small and
large intestines (Figures 15 and 16).
a frank cancer might be easy to detect (as it would be by any technique),
28,29
Under these circumstances, while
Lower Gastrointestinal Tract 99
Figure 16. Colon, OCT. Again, insufficient contact for uniform visualizationof mucosa. Normal crypts
only become slightly apparent toward the area of iatrogenic compression. Sh: Sheath (of balloon).
M: Mucosa. SubM: Submucosa. MP: Muscularis propria.
29
more subtle changes, such as focal dysplasia and inflammation, would not
be. For completeness, OCT is an interference technique that translates a
near-infrared signal reflected from tissue in conjunction with a reference
arm to produce high-resolution cross-sectionalimages of tissue microstructure. In principle, the technique is akin to ultrasonography.
2
Cross-sectional
images are obtained at a frequency of up to 30 frames per second to depths
of up to 2 mm (generally mucosa and submucosa) and resolutions from
10 to 20 µm.
nel of conventional endoscopes.
29
OCT devices can be passed through the accessory chan-
30
As elsewhere, standard OCT techniques
cannot visualize subcellular details and do not rely on contrast administration. Areas of active investigation via OCT in the lower GI tract include
celiac disease, epithelial dysplasia/neoplasia detection, and post-radiation
monitoring for re-epithelialization.
31