Page 579 - The Veterinary Laboratory and Field Manual 3rd Edition

516 Samuel Sharpe

Histopathology Immunohistochemistry (IHC) and in situ
hybridization (ISH) are both techniques which
Histopathology is the study of microscopic can be performed on tissue sections which uti-
morbid anatomy and relies on identification lize the specificity of antibody binding to detect
of characteristic microscopic changes associ- protein antigen (IHC) or nucleic acid (ISH) in
ated with various pathological processes. This tissues. IHC is commonly used in diagnostic labs
allows further investigation of the underlying to detect antigens in tissue section. In surgical
cause of lesions recognized grossly. In some pathology expression patterns of cellular anti-
cases, microscopic changes will be pathogno- gens in tumours can be used to give accurate
monic and definitive diagnosis can be made from diagnosis and prognostic information. IHC can
examination of the tissue section. More com- also be used to detect infectious agents in tis-
monly changes will be suggestive of a pathologic sues.
process allowing further refinement of the differ- All histochemical and the majority of IHC
ential diagnosis list and selection of appropriate and ISH techniques can be performed on tissue
ancillary testing. routinely fixed in 10% NBF. If you have ques-
tions regarding sampling and tissue preservation
for a specific test please contact your diagnostic
Histochemical staining
laboratory.
The process of thinly sectioning processed tis-
sue and staining with a variety of dyes and other Sampling technique and preservation
chemicals for microscopic examination has
changed little since its invention several centu- Tissue samples for histology should be taken
ries ago. into a fixative solution the purpose of which is to
Routine examination is performed on tis- stabilize proteins in the sample and inhibit deg-
sue sections stained with Haematoxylin and radation prior to processing for histopathology.
Eosin (H+E). H+E differentially stains cellu- In addition, fixation increases tissue stiffness
lar elements (for example, nuclei stain blue or and makes cutting of tissue sections easier.
‘basophilic’, cytoplasm stains pink or ‘eosino- Commonly used fixatives act by either reduc-
philic’) allowing recognition and interpretation ing solubility and disrupting tertiary structure of
by trained personnel. In addition, cells or tissue proteins (so-called denaturing fixatives, usually
elements affected by a specific disease process ethanol or methanol) or by stabilizing proteins
will display stereotypical colour changes aiding by creating covalent bonds within and between
in identification of that process. For instance, an protein molecules (so-called cross-linking fixa-
acutely necrotic neuron may appear shrunken, tives, usually formaldehyde).
with a more densely staining and darker nucleus Ten per cent neutral buffered formalin (10%
and more eosinophilic cytoplasm. NBF) is the most widely used fixative in anatom-
Other histochemical (‘special’) stains can be ical pathology and can be purchased ready-made
used to highlight tissue elements (for example, in liquid form or as a dry powder ready to be
collagen, GAGs, elastin, reticulin), intra- or reconstituted.
extra-cellular accumulations (for example, It is strongly recommended to use ready-
glycogen, mucin), pigments (for example, hemo- made 10% NBF if available, as formalin powder
siderin, melanin, bile, haemoglobin), minerals is an irritant to mucous membranes and can be
(iron, copper, lead) or microorganisms (bacteria, hazardous to work with. Additionally, it is very
protozoa, fungi). important to correctly buffer the pH of the solu-

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