Clinical Biochemistry of Domestic Animals (Sixth Edition) - UMK ...

V. Immunohistochemistry/Immunocytochemistry
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E. Image Analysis
Obtaining morphometric and densitometric measurements
from individual nuclei and providing information
about ploidy, S-phase fraction, and nuclear area, are possible
with image analysis of cytocentrifuge preparations of
Feulgen-stained nuclei extracted from formalin-fixed, paraffin-embedded
tissues. Morphometric measurements can
also be obtained from hematoxylin and eosin (H & E) and
panoptic-stained cytology specimens. Nuclear morphometry
and ploidy may aid in the classification of canine mast
cell tumors ( Strefezzi Rde et al. , 2003 ), distinguish benign
from malignant melanocytic tumors in dogs and cats ( Roels
et al. , 2000b ), and predict the outcome of feline mammary
carcinomas ( De Vico and Maiolino, 1997 ).
F. Apoptosis
Apoptosis is an energy-dependent process that may be considered
a “ programmed ” form of cell death, distinct from the
“ accidental ” cell death of necrosis. During apoptosis, there
is activation of endogenous nucleases, which create oligonucleosomal
fragments that have a characteristic orderly ladder
appearance when separated by electrophoresis, compared with
the smeared appearance of DNA degraded during necrosis.
Cells undergoing apoptosis have characteristic morphological
features including condensation of nuclear heterochromatin
and resultant crescent apposed to the nuclear membrane,
cell shrinkage, cytoplasmic condensation, and bud formation
at the cell membrane, which may condense into “ apoptotic
bodies. ” Another characteristic of apoptosis is the absence of
inflammation, which is typically present with necrosis.
Although features of apoptosis may be identified with
light microscopy of H & E-stained cells, electron microscopy
or fluorescence microscopy with dyes such as propidium
iodine or acridine orange may also be useful.
The apoptotic index predicts the initial relapse-free survival
in dogs with lymphoma, but not its overall survival
( Phillips et al. , 2000 ). However, the apoptotic index does
not appear to correlate with survival in feline mammary
tumors ( Sarli et al. , 2003 ).
V. IMMUNOHISTOCHEMISTRY/
IMMUNOCYTOCHEMISTRY
Immunohistochemistry is now a well-accepted and routinely
applied method in most every veterinary diagnostic
laboratory. Similar techniques have been applied to cytology
specimens with good result. These methods involve
the use of antibodies, which bind specific cellular components
representing specific markers of the cell type of
origin. By linking the antibodies to a dye, the immunoreactivity
between the tissue specimens and antibodies can be
visualized with the light microscope.
The use of immunohistochemistry and immunocytochemistry
has advanced the understanding of tumor differentiation
and allowed for improved tumor typing.
Immunohistochemistry facilitates the determination of histogenesis
of many tumors that might otherwise be classified
as undifferentiated on the basis of light microscopy of
routine H & E stained tissues. Although the antibodies used
for immunohistochemical staining do not recognize unique
attributes of the tumor nor do they differentiate benign from
malignant cells per se, the accurate determination of histogenesis
may allow the clinician to choose appropriate treatment
and formulate an accurate estimate of the prognosis.
It is beyond the scope of this chapter to exhaustively review
all immunohistochemical and immunocytochemical markers
useful for veterinary cancer diagnosis. What follows
are examples of those markers that well illustrate the use of
these techniques as tumor markers.
A. Epithelium
It is often difficult to distinguish carcinoma for other poorly
differentiated or undifferentiated neoplasms. The immunohistochemistry
(IHC) markers that are commonly used to characterize
tumors of epithelial origin are antibodies directed
against the cytokeratin intermediate filaments. The cytokeratin
proteins are unique among the intermediate filaments of
the cytoskeleton because of their high degree of diversity
in polypeptide units and the fact that cytokeratin intermediate
filaments are highly correlated with the degree of tissue
differentiation. Therefore, identification of specific cytokeratins
can be useful for confirming epithelial histogenesis
and distinguishing glandular from squamous differentiation
independent of other morphological features. As epithelial
tumors progress from dysplastic epithelium to carcinoma in
situ to invasive squamous cell carcinoma, there is a concurrent
decrease in expression of both high- and low-molecular-weight
cytokeratins. Although a majority of canine and
feline carcinomas will have immunoreactivity of at least
some neoplastic cells for cytokeratins, the use of a panel of
anticytokeratin antibodies, to include both high- and lowmolecular-weight
keratins, may improve the sensitivity of the
immunohistochemistry. It is noteworthy that many studies of
cytokeratin intermediate filaments in domestic animal tumors
have used antibodies directed against human cytokeratins,
and there are clear differences in the observed immunoreactivity
among species for many of these antibodies.
Specific cytokeratins have been proposed as diagnostic
markers for some tumors owing to a change in cytokeratin
expression as a consequence of tumorigenesis. For
example, poorly differentiated prostatic carcinoma in dogs
has positive immunoreactivity for cytokeratin AE1/AE3
( Grieco et al. , 2003 ), yet cytokeratin 7 is not sufficient to
distinguish prostatic from transition cell carcinomas in
dogs ( LeRoy et al. , 2004 ). Various canine skin tumors may