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
190 J. M. Eschbacher
et al.
show tissue properties at the microscopic level, providing guidance for
tissue biopsy and potentialtumor margin interrogation for extending or optimizing tumor resection. In essence, CLE provides a digital optical biopsy
“on-the-fly” during surgery.
In the central nervous system (CNS) setting, fluorescein sodium, a
contrast agent approved by the U.S. Food and Drug Administration, is
administered intravenously 2–5 min before intraoperative imaging. Fluorescein leakage through vessels provides a bright background that contrasts
with tumor cells and structures. Histopathologic tissue features are most
prominent in the tumor and peritumoral regions, where permeable vessels
allow for fluorescein leakage into tissue. Tissue structures and cells generally appear as dark or silhouetted objects against a bright background. With
optimal images, nuclear detail may be appreciated, but cell shape and size,
as well as degree of cellularity and tumor growth patterns, are identifiable.
One barrier to CNS adaptation of CLE techniques has been that many
topical dyes, such as acriflavine, cannot be applied to the brain surface due
to toxicity. However, fluorescin sodium could be injected intravenously; it
is used for fluorescein angiography, which has been used in vascularneurosurgery and is performed by ophthalmologists during evaluation for various
2
eye diseases, including glaucoma.
Fluorescein readily crosses the bloodbrain barrier where initial leakage into the tumor and peritumoral tissue
interstitial space provides contrast for visualization of tumor architecture
and cell morphology. Cell bodies, vascular structures, and tissue architecture appear dark, contrasted against a bright fluorescent background.
As a novel and evolving imaging modality, CLE imaging has been
described in a growing number of available reports on the in vivo application in the CNS and, specifically, in the first feasibility studies of the clinical
grade system.
1,3–10
Given its cell resolution imaging capability, this technique has the potential to greatly impact howwe approach the intraoperative
diagnosis and resection of CNS tumors. In the clinical setting, CLE can be
used intraoperatively to guide tissue diagnosis and potentially replace or
augment the historical gold standard for neurosurgical intraoperative diagnosis: the frozen section method.
Frozen section analysis is routinely used during brain and spinal cord
surgery.
11
Indications include obtaining a preliminary diagnosis, which
may guide surgical strategy, and evaluating tumor margins. Frozen section
1
Central Nervous System 191
evaluation requires approximately20 min, during which tissue is frozen and
cut with a specialized instrument (a cryostat). Slides with frozen section
material are stained and examined by a pathologist. Frozen section consultation, althoughhelpful to the surgeon as an intraoperative preliminary diagnosis, does carry distinct disadvantages that include misdiagnoses related
to sampling and processing errors. Intraoperative missampling of tissue can
lead to undergrading of tumors, one of the most commonly reported sources
of error in neuropathologyfrozen section analysis.
12–14
Frozen section artifact, prevalentin edematous brain tissue, can also complicate interpretation
and lead to multiple additional biopsies for diagnosis, which can convey
additional risk to the anaesthetized patient. Pathology laboratory operating
costs and risk to personnel (e.g. exposure to sharp instruments and infectious agents) must also be considered in the frozen section setting.
CLE also allows for optical sectioning deep into tissue, providing
images at depths not obtainable with standard frozen section techniques.
Although imaging depths depend on the optical scattering properties of the
tissue and the wavelengths of excitation and emitted light used, a 488 nm
excitation light allows for interrogation of planes up to 30µm deep into
brain or tumor tissue. These tissue depths provide images at more than 6
times deeper than standard 4- to 5-µm frozen tissue sections. Because of
its rapidity, its non-tissue-consuming nature, and its potential image depth,
in vivo CLE has the potential to be used as a clinical diagnostic tool to
replace or augment frozen section techniques in the CNS setting. CLE can
provide a rapid intraoperative method for biopsy analysis while minimizing
errors and risks inherent to routine frozen section analysis.
Technique
Currently, in vivo CLE imaging of brain lesions is in the nascent phase and
not widely available for use in the clinical setting. Carl Zeiss Meditec, AG
(Oberkochen, Germany), working with Optiscan (Melbourne, Australia),
has produced two CLE microscopes dedicated to in vivo CNS imaging
(Optiscan 5.1), and a US Food and Drug Administration-approved clinicalgrade generation endomicroscope (CONVIVO), which was approved for in
vivo use in 2018 (Figure 1). The Zeiss microscope functions as a miniature
192 J. M. Eschbacher
Figure 1. CONVIVO imaging workstation. Reproduced with permission from Carl Zeiss Meditec, AG,
Oberkochen, Germany.
et al.
handheld confocal microscope that delivers laser light (blue, 488 nm wavelength) to the tissue via an optical fiber. Fluorescence is collected by the
lens system and focused onto the tip of the optical fiber, which acts as
a confocal pinhole. A foot pedal is used to adjust Z depth and capture
images. Altering the position of the focal plane provides an imaging depth
of up to 500 µm. Confocal image data are collected at a minimum of
0.7 frames/second (1920 × 1200 pixels) to a maximum of 4 frames/second
(1024 × 128 pixels). The rigid endoscopic probe has a diameter of 4mm
(Figure 2). Images are displayed as grayscale or pseudocolored pictures on
a video console mounted on a movable cart. During surgery, the handheld
probe is dressed with a sterile single-use cover and placed on the instrument table, similar to suction or bipolar coagulation devices. A navigation
tracking frame can be attached to the CLE probe to correlate the location of
the optical biopsy to magnetic resonance or computed tomography imaging
data. Imaging of the tissue is performed by the neurosurgeon when necessary in a freehand fashion or by fixing the probe in a rigid retractor arm with
further positioning against the tissue. The surface of the tumor is washed
with sterile saline as needed to reduce the burden of erythrocytes. Imaging
Central Nervous System 193
Figure 2. The endoscopic probe for tissue imaging. Reproduced with permission from Carl Zeiss
Meditec, AG, Oberkochen, Germany.
time typically varies between 2 and 10 min. After each site is studied, the
location is marked on the image guidance system (StealthStation TREON,
Medtronic, Inc., Dublin, Ireland).
Histopathology of Optical Images
Normal brain, dura, blood vessels, and blood
Normal structures are evident in non-neoplastic brain tissue; however,
because of a lack of edema and thus contrast leakage, architectural and cellular structures in normal nonedematous brain may appear faint (Figure 3).
The background neuropil typically exhibits a cobweb-like architecture
consisting of numerous strands of haphazardly arranged thin fibers, consistent with axonal and astrocytic processes. Neurons or glia appear as
intermediate-sized cell bodies. Corpora amylacea appear as spherical bodies. Dural tissue is generally hypocellular with scattered dark wavy fibers.
Cortical surface vessels are tortuous and bright with fluorescein, whereas
intraparenchymalvessels appear thin and branching. Erythrocytesare round
and often refractile, and they have a characteristic 7µm size. Their disclike shapes may contaminate images but are distinguishable from other
194 J. M. Eschbacher
Figure 3. Confocal laser endomicroscopy images of normal brain adjacent to low-grade gliomas
show axons and vessels. Reproduced with permission from B arrow Neurological Institute, Phoenix,
Arizona.
et al.
tissue elements. Time-series imaging allows for easy differentiation of erythrocytes from other stationary cells by observation of their movements
and characteristic well-delineated regular and uniform oval-round shape.
Time-series imaging also allows tracking of erythrocytes and other cells
within the blood vessels, with easy discrimination of flow even within
capillaries.
4
CNS Tumors
Both intra-axial and extra-axial CNS tumors viewed with CLE show cellular and architectural characteristics that closely mirror their described
pathology using standard staining.
fibrous, meningothelial,chordoid, and secretory growth patterns (Figure 4).
Classical features such as collagen (Figure 5) and psammoma bodies
(Figure 4) a re visible. Cell morphology is often evident, with many cells
1,6, 7
Meningioma subtypes demonstrate
Central Nervous System 195
(a)(
)
b
Figure 4. (a) Hematoxylin and eosin–stained and (b) confocal laser endomicroscopy images from
a meningothelial meningioma show similar architectural patterns. Arrowheads indicate psammoma
bodies. Adapted from Eschbacher et al. [7].
(a) (b)
Figure 5. (a) Hematoxylin and eosin–stained and (b) confocal laser endomicroscopy images from
a transitional meningioma show collections of fibers consistent with collagen (arrowheads). Adapted
from Eschbacher et al. [7].
showing dark intracytoplasmic structures c onsistent with nuclei. Gliomas
can demonstrate areas of hypercellularity and necrosis (Figure 6). Cellular
atypia is appreciable.Infiltrating tumor can oftenbe appreciated, which may
have ramifications for surgical margin resection (Figure 7). Features such
as ependymal perivascular pseudorosettes can be recognized (Figure 8).
196 J. M. Eschbacher
(a)(
)
(a)(
)
Figure 6. (a) Hematoxylin and eosin–stained and (b) confocal laser endomicroscopy images from
an oligodendroglioma (World Health Organization grade 3) show a hypercellular tumor with similar
architecture and nuclear pleomorphism. Arrows denote vessels. Adapted from Eschbacher et al. [7]
et al.
b
b
Figure 7. An oligodendroglioma viewed on (a) hematoxylin and eosin–stained and (b) confocal laser
endomicroscopy images shows similar cellularity,architecture, and atypia. Adapted from Eschbacher
et al. [7].
Other tumor types show cytoarchitectural features that resemble those seen
on hematoxylin and eosin–stained sections, including hemangioblastomas
(Figure 9), schwannomas, and metastatic tumors. Non-neoplastic lesions
also recapitulate standard histology, as seen with hemangiomas and treated
tumors (Figure 10). Overall, in most studied tumor types to date, nucleoli
are only occasionallyvisible. Mitosis can be difficultto appreciate, although
more study is needed to evaluate its presence.
Central Nervous System 197
(a)(
)
(a)(
)
b
Figure 8. Perivascular pseudorosettes (braces) in an ependymoma shown on (a) hematoxylin and
eosin–stained and (b) confocal laser endomicroscopy images. Adapted from Eschbacher et al. [7].
b
Figure 9. (a) Hematoxylin and eosin–stained and (b) confocal laser endomicroscopy images from a
hemangioblastoma show similar growth patterns. Adapted from Eschbacher et al. [7].
Artifacts
Two common sources of artifacts that compromise image interpretation
are erythrocyte contamination of the imaging field and motion artifacts.
Erythrocyte c ontamination appears as sheets of small disc-likecells moving
across the field (Figure 11). Flushing the imaging field with sterile saline
can be helpful in washing away the erythrocytes and exposing underlying
tissue for imaging. Motion artifact (Figure 12) appears as irregular waves
198 J. M. Eschbacher
(a)(
)
Figure 10. (a) Confocal laser endomicroscopy shows overall a lack of contrast consistent with
necrosis seen on the (b) hematoxylinand eosin–stained image in a treated glioblastoma. Reproduced
with permission from Barrow Neurological Institute, Phoenix, Arizona.
et al.
b
Figure 11. Sheets of erythrocytes contaminate a surgical field during imaging with confocal laser
endomicroscopy. Reproduced with permission from Barrow Neurological Institute, Phoenix, Arizona.
across the screen. It can be minimized by maintaining a steady hand during
imaging or bracing the wrist or hand against a solid object while holding
the probe. Instruments devised to stabilize the probe may also be used but
Central Nervous System 199
Figure 12. Motion artifact obscures images of a glioblastoma obtained with confocal laser endomicroscopy. Reproduced with permission from Barrow Neurological Institute, Phoenix, Arizona.
are not recommended, because they may damage the probe, which was
originally designed to allow for free-hand scanning during surgery.
Limitations
More studies are needed to evaluate the sensitivity and specificity of in vivo
CLE in inflammatory, infectious, and autoimmune disorders (e.g. abscess
and multiple sclerosis), as most images collected to date have been obtained
in the tumor setting. Specifically, non-tumor disease processes should be
contrasted histopathologically with known tumor features observed on CLE
in order to learn how these disease entities present and to avoid misdiagnosis
and potential harm to the patient. Some histologic features (e.g. nuclear
hyperchromasia, mitotic activity, and prominent nucleoli) have not been
well documented with fluorescein. In such instances, ex vivo rapid CLE of
acriflavine- or acridine orange–stained fresh biopsy samples may provide
complementary images.