Immunohistochehemical Technology

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Suggested Guidelines for Immunohistochemical
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J VET Diagn Invest
Czub, Fabio Del Piero, Sharon Dial, E. J. Ehrhart, Tanya Graham, Lisa Manning, Daniel Paulsen and Victor E. Valli
José A. Ramos-Vara, Matti Kiupel, Timothy Baszler, Laura Bliven, Bruce Brodersen, Brian Chelack, Keith West, Stefanie
Suggested Guidelines for Immunohistochemical Techniques in Veterinary Diagnostic Laboratories
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3. Suggested guidelines for immunohistochemical techniques in veterinary
diagnostic laboratories
José A. Ramos-Vara,1
Matti Kiupel,2
Timothy Baszler, Laura Bliven, Bruce Brodersen,
Brian Chelack, Stefanie Czub, Fabio Del Piero, Sharon Dial, E. J. Ehrhart, Tanya Graham,
Lisa Manning, Daniel Paulsen, Victor E. Valli, Keith West
Abstract. This document is the consensus of the American Association of Veterinary Laboratory
Diagnosticians (AAVLD) Subcommittee on Standardization of Immunohistochemistry on a set of guidelines
for immunohistochemistry (IHC) testing in veterinary laboratories. Immunohistochemistry is a powerful ancillary
methodology frequently used in many veterinary laboratories for both diagnostic and research purposes. However,
neither standardization nor validation of IHC tests has been completely achieved in veterinary medicine. This
document addresses both issues. Topics covered include antibody selection, fixation, antigen retrieval, antibody
incubation, antibody dilutions, tissue and reagent controls, buffers, and detection systems. The validation of an
IHC test is addressed for both infectious diseases and neoplastic processes. In addition, storage and handling of
IHC reagents, interpretation, quality control and assurance, and troubleshooting are also discussed. Proper
standardization and validation of IHC will improve the quality of diagnostics in veterinary laboratories.
Key words: Animal diseases; diagnostic immunohistochemistry; standardization; validation.
Introduction
Morphological diagnosis in veterinary medicine has
classically relied mostly on routine stains such as
hematoxylin and eosin and, less commonly, on other
histochemical stains. However, the level of specializa-
tion in veterinary practice demands more accurate
diagnosis, particularly for tumors. Immunohistochem-
istry (IHC) has been proven to be one of the most
important ancillary techniques in the characterization
of neoplastic diseases in humans and has become
equally important in veterinary medicine, as oncolo-
gists demand more specific diagnoses. The number of
immunohistochemical tests offered by veterinary diag-
nostic laboratories for the diagnosis of infectious and
neoplastic diseases in frozen or formalin-fixed, paraf-
fin-embedded (FFPE) tissues has increased exponen-
tially in the last decade. Immunohistochemistry has
been proven as a highly specific and sensitive diagnostic
method that is especially advantageous as a diagnostic
tool for infectious diseases. In some cases, IHC is
considered the gold standard technique to which others
are compared (e.g., prion diseases). In comparison with
other diagnostic tests, IHC allows colocalization of an
antigen with a lesion, thereby dramatically increasing
diagnostic accuracy and understanding of pathogene-
sis. Numerous infectious agents and cell types can be
identified with IHC in a wide variety of animal species,
especially since the advent of antigen retrieval methods
using heat.62
However, there are no guidelines for
standardization and no general agreement on the use of
controls or the validation of a test in veterinary
diagnostic IHC. The purpose of the present article is
to provide guidelines for IHC in the veterinary
diagnostic laboratory, and is not intended as manda-
tory requirements for laboratory accreditation. The
article briefly reviews the technical aspects of IHC and
provides guidance for standardization, test validation,
controls, storage and handling, interpretation and
From the Animal Disease Diagnostic Laboratory, Purdue
University, West Lafayette, IN (Ramos-Vara), Diagnostic Center
for Population and Animal Health, Michigan State University, East
Lansing, MI (Kiupel), Washington Animal Disease Diagnostic
Laboratory, Washington State University, Pullman, WA (Baszler),
Marshfield Clinic Laboratories Veterinary Services, Marshfield, WI
(Bliven), Veterinary Diagnostic Laboratory, University of Ne-
braska, Lincoln, NE (Brodersen), Prairie Diagnostic Services Inc.,
Saskatoon, Saskatchewan, Canada (Chelack, West), National
Centre for Foreign Animal Diseases, Canadian Food Inspection
Agency, Winnipeg, Canada (Czub), Department of Pathobiology
and Clinical Studies, New Bolton Center, University of Pennsylva-
nia, Kennett Square, PA (Del Piero), Department of Veterinary
Science and Microbiology, University of Arizona, Tucson, AZ
(Dial), Department of Microbiology, Immunology, and Pathology,
Colorado State University, Fort Collins, CO (Ehrhart), Animal
Disease Research and Diagnostic Laboratory, South Dakota State
University, Brookings, SD (Graham), National Centre for Foreign
Animal Diseases, Canadian Food Inspection Agency (Manning),
Department of Pathobiological Sciences, Louisiana State Univer-
sity, Baton Rouge, LA (Paulsen), and Department of Pathobiology,
University of Illinois, Champaign, IL (Valli).
1
Corresponding Author: José A. Ramos-Vara, Animal Disease
Diagnostic Laboratory, Purdue University, 406 South University,
West Lafayette, IN 47907. ramosja@purdue.edu
2
Dr. Kiupel is Chair of the AAVLD Subcommittee on
Standardization of IHC.
J Vet Diagn Invest 20:393–413 (2008)
393

4. quality control and assurance, as well as general
troubleshooting protocols.
Overview of the immunohistochemical test
The IHC technique is a combination of immuno-
logic and chemical reactions visualized with a photonic
microscope. The IHC technique can be divided into 4
main steps (Fig. 1). Step 1 includes all preimmunologic
procedures done before incubation with the primary
antibody. These procedures include deparaffinization
and hydration of tissue sections, blocking nonspecific
bindings, blocking endogenous activities (e.g., perox-
idase, phosphatase, biotin), and antigen retrieval. Step
2 includes all the immunologic reactions between the
primary antibody and tissue antigens, the primary
antibody and secondary antibody and some additional
chemical reactions necessary to bind the reporter
molecule (label) such as peroxidase or alkaline
phosphatase to the preformed immune complex. Step
3 includes all the procedures necessary to visualize the
antigen-antibody binding. In IHC, this is achieved with
a chemical reaction in which the label reacts with its
substrate and a chromogen to produce a colored
reaction product. Then, immunolabeled tissue sections
are counterstained and coverslipped. Step 4 includes
interpretation and reporting of the IHC results.
Standardization of a new test
Standardization determines optimal conditions
(e.g., incubation time, incubation temperature, dilu-
tions, controls, buffers, detection system) to ensure
that the selected antibody reacts with the expected
antigen (Fig. 2). There is a lack of standardization
among different veterinary laboratories, despite the
widespread use of IHC.47
Contrary to the situation in
human IHC, a major problem in veterinary IHC is
the reduced availability of high-quality antibodies
that are specific for infectious agents of veterinary
importance, and cell markers specific for small and
large animal species. Especially in tumor diagnostics,
most laboratories use batteries of antibodies devel-
oped for human tissues that are often insufficiently
validated for animal species. With the exception of
the surveillance program for prion diseases (e.g.,
scrapie, chronic wasting disease), neither the proto-
cols nor the primary antibodies are uniform among
diagnostic laboratories.
There is also little guidance or regulation for the
actual standardization of specific IHC tests in human
medicine although a consensus on guidelines for
quality assurance for IHC has been published by the
Clinical and Laboratory Standards Institute (for-
merly National Committee on Clinical Laboratory
Standards, 1999).1
The Food and Drug Administra-
tion (FDA) has classified most IHC reagents and kits
as ‘‘Analyte Specific Reagents,’’ thereby exempting
them from premarket notification (FDA regulations
classify devices depending upon the degree of
regulation necessary to provide reasonable assurance
of their safety and effectiveness). This ruling was
Figure 1. Overview of immunohistochemical test. The immunohistochemistry technique can be divided into 4 main sections:
pretreatments (stage 1), immunologic and histochemical reactions (stage 2), visualization of the immunologic reaction (stage 3), and
interpretation and report generation (stage 4).
394 Ramos-Vara et al.

5. based on the assumption that most IHC tests are part
of the pathologist’s report and, as such, will not be
reported independently. For antibodies used as stand-
alone tests in human pathology, premarket notifica-
tion and specific FDA approval are required. For
stand-alone tests in veterinary IHC, premarket
notification by regulatory agencies (U.S. Department
of Agriculture [USDA]) is not required. However, for
certain animal diseases (e.g., scrapie, chronic wasting
disease, Bovine viral diarrhea virus), IHC results are
commonly reported as stand-alone tests, so standard-
ization of these tests among various laboratories is
urgently needed. The main goal of standardization in
IHC is to obtain reproducible and consistent results
within each laboratory and comparable results among
different laboratories even when using different
procedures. In this review, each of the main
components of an IHC test, including primary
antibody, fixation, antigen retrieval, pretreatments,
and detection systems, are addressed.
Primary antibody characterization
Monoclonal and polyclonal antibodies: sensitivity
and specificity. Monoclonal antibodies (mAb)
derived from a single B-cell clone and produced by
hybridoma technique provide excellent specificity
because the antibody binds to a single epitope on 1
antigen.43
most mAb are of mouse origin, although
rabbit mAb are also commercially available.56,69
Polyclonal antibodies (pAb) contain antibodies to a
range of antigens and thus may cause greater
nonspecific background staining and be less specific
than mAb. Subjecting pAb to multiple adsorption
protocols for a variety of antigens will increase their
specificity. For both mAb and pAb, the diagnostic
specificity of an antibody detecting infectious agents
is the positive immunoreactivity against the targeted
agent only and lack of immunostaining in tissues
infected with other agents. The diagnostic specificity
of an antibody detecting cellular/tumor markers is the
expected presence or absence of immunostaining in
certain cell types, tissues, or tumors in multitissue
control blocks (see Controls section).64
Analytical
sensitivity is defined as weak (positive) staining in the
presence of the least amount of antigen. Polyclonal
antibodies may be more sensitive than mAb because
they can bind several different epitopes on a single
antigen.64
Diagnostic sensitivity, defined as the
proportion of known positive samples that test
positive,65
is established by comparing the test
results on FFPE tissue with results using another
antibody that has been validated for the same analyte
or using a non-IHC method such as culture or
polymerase chain reaction (PCR).
Cellular markers.—With the exception of CD (clu-
ster of differentiation) markers, there are few cellular
antigens in animals for which species-specific
antibodies have been developed, and even fewer are
Figure 2. Steps to consider during the standardization of a new immunohistochemical test. Five main factors are involved in
immunohistochemistry standardization: animal tissue, fixation, primary antibody, antigen retrieval, and detection systems.
Immunohistochemical techniques in veterinary diagnostic laboratories 395

6. detectable in FFPE tissues. Most antibodies to
cellular antigens used in veterinary IHC laboratories
have been developed against human or rodent
antigens, and characterization data for their
specified use should be available. Cellular markers
should demonstrate reactivity with the appropriate
molecular weight antigen in Western blots (WB);
however, WB immunoreactivity does not necessarily
imply or predict immunoreactivity in FFPE tissues.
Interpretation of IHC results requires familiarity
with the expected pattern of immunoreactivity based
on location of the antigen (e.g., nuclear, membrane,
or cytoplasmic staining; Fig. 3).76
For example,
staining for cytokeratins and vimentin should be
cytoplasmic, not nuclear; staining for thyroid tran-
scription factor 1 (TTF 1) in lung or thyroid tumors is
nuclear, not cytoplasmic.48,51
In other words, the
presence of staining does not always indicate a
positive reaction. In most cases, the degree of specific
staining should vary between cells and in some cases
within different cell compartments; such cellular
distribution may have prognostic significance in
certain tumors.33
Infectious agents.—For viral agents, positive stain-
ing should be detected in the appropriate tissues, cell
types, and cellular location (if known), in association
with typical lesions. For protozoal and bacterial
pathogens, the morphology and location of the
stained organisms can provide some additional
information.
Fixation
The ultimate goal of fixation is to preserve cells and
tissues, to prevent autolysis, and to preserve antige-
nicity. Unfortunately, no fixative can optimally fulfill
all these requirements.43
The most common type of
fixation in diagnostic IHC is chemical, specifically
with formaldehyde. The time lapse between the death
of the animal and the collection of tissues for IHC is
usually critical, and prompt transfer into fixative is
desired. Enzyme-rich tissues, such as intestine or
pancreas, autolyze rapidly. Protozoa and fungi may
be more resistant to autolysis than viruses. In general,
samples to be fixed should not be thicker than 0.5 cm,
and the ratio of fixative to sample should be at least
10 to 1. Factors influencing the quality of fixation
include type of fixative, fixative pH and buffers,
fixative concentration, fixative osmolality, fixation
time, fixation temperature, fixative additives, and the
use of additional post fixation procedures (e.g.,
decalcification).
Coagulative fixation. Coagulative fixatives are
organic and nonorganic solutions that coagulate
proteins and render them insoluble. The fixation
process removes and replaces free water, thereby
destabilizing hydrophobic and hydrophilic bonding
(hydrophobic areas are released from the repulsion of
water, and occupy a greater area). This causes a
disruption of tertiary protein structure and results in a
partially reversed (‘‘less folded’’) protein structure.43
The disruption of tertiary structures also changes
physical properties, and proteins that are normally
soluble in aqueous solution become insoluble.
Coagulative fixation maintains tissue structure at
the light microscopic level fairly well, but results in
cytoplasmic flocculation as well as poor preservation
of mitochondria and secretory granules.43
The most
common types of coagulative fixatives are dehydrants
(alcohols and acetone) and strong acids (picric acid,
trichloroacetic acid).
Cross-linking fixation. Cross-linking fixatives
form cross-links within and between proteins, within
and between nucleic acids, and between nucleic acids
and proteins. The most common cross-linking
fixatives are aldehydes (formaldehyde,
glutaraldehyde, chloral hydrate, glyoxal), with
neutral buffered formaldehyde most frequently used
in routine histopathology. Formalin fixation
produces methylene bridges between free amino
groups and other functional groups of proteins,
resulting in conformational changes in the tertiary and
quaternary structures within proteins and cross-linking
between tissue proteins, thus modifying the epitopes
recognized by antibodies.3,15,21,22,43,71
However, it needs
to be emphasized that adequate fixation is essential in
IHC. Underfixation of tissues for IHC testing is one of
the major problems in human pathology.15
Tissues that
are poorly fixed in formalin will be subjected
to coagulative fixation (alcohol) during tissue
processing, resulting in inadequate antigen retrieval or
unexpected antigen detection. Underfixed tissues
cannot withstand harsh retrieval methods and are
easily damaged, with subsequent loss of antigenicity.54
Antigen retrieval
Antigen retrieval (AR) is intended to reverse the
detrimental effects of fixation. As mentioned, one
of the main effects of formalin fixation is
conformational changes in epitopes, but loss of
electrostatic charges may occur during fixation and
have an effect on antigen detection.16
The exact
mechanism by which AR works on formalin-fixed
tissues is not clear. A variety of pathways may
contribute to its success, including the breaking of
cross-linkages, the extraction of diffusible blocking
proteins, the precipitation of proteins, the hydrolysis
of Schiff’s bases, calcium chelation, paraffin removal,
and the rehydration of tissue, resulting in better
penetration of antibody and increased accessibility to
396 Ramos-Vara et al.

7. Figure 3. Cellular location of antigens. Antigens can be located in one or multiple cell compartments. Knowledge of the expected
location of a particular antigen is essential to an adequate interpretation of the immunohistochemistry results. A, testis of a dog with
Sertoli cell tumor. Neoplastic cells have strong nuclear staining for GATA-3, which is the expected location for this antigen. Panels B–D
are examples of KIT distribution in mast cell tumors. KIT cellular distribution has been linked to mast cell tumor prognosis in dogs but
not in other species. B, skin from a horse with mast cell tumor. Neoplastic cells depict only membrane staining for KIT. C, skin from a
dog with mast cell tumor. The majority of cells show diffuse cytoplasmic staining for KIT. D, skin from a horse with mast cell tumor. A
mixed staining pattern including cytoplasmic membrane, cytoplasm, and paranuclear staining is observed in many cells.
Immunoperoxidase-3,39-diaminobenzidine (DAB). Bar 5 20 mm.
Immunohistochemical techniques in veterinary diagnostic laboratories 397

8. antigen.37,62
Longer fixation times are thought to
decrease immunoreactivity and might require longer,
harsher AR methods, increased antibody
concentrations or longer primary antibody
incubation times to achieve a similar degree of
immunoreactivity. However, with the advent of
heat-induced AR, retrieval of antigens in overfixed
tissues can still be achieved in many instances.45,48,51
In a recent study using 30 antibodies targeting cellular
antigens in different cell compartments and infectious
agents, only those to Canine parvovirus, Felid
herpesvirus, and cytokeratin 7 had a significant
reduction in the signal after several weeks of fixation
(Webster J, Miller M, Ramos-Vara J: 2007, Proc
AAVLD, p. 86). Background staining caused by harsh
AR methods is not uncommon, and sometimes
precludes diagnostic evaluation of the staining. Three
main AR methods are currently used in FFPE tissues:
heat-induced AR, enzymatic digestion, and detergent-
induced AR (Fig. 4). For a practical approach to
antigen retrieval, see the next section (‘‘Practical
approach to standardization of a new antibody’’).
Heat-induced antigen retrieval
The success of heat-induced AR is probably the
result of reversal of formalin-induced chemical
modifications in protein structure. The source of
heat is not critical to the outcome, but a matter of
convenience. Various types of equipment may be used
including a de-cloaker (commercial pressure cooker
with electronic controls for temperature and time),
vegetable steamer, microwave oven, or pressure
cooker.7,20,32,41,63
Many veterinary diagnostic
laboratories use a steamer and/or de-cloaker. The
advantage of a de-cloaker over other heating devices
Figure 4. Effects of different antigen retrieval (AR) in the immunohistochemical reaction. Small intestine, dog. A, this section was
treated with proteinase K as AR resulting in no staining. B, the staining intensity in the muscle layer (m) is very strong but also the
background produced in the submucosa(s). A combined (proteinase K and heat-induced AR citrate) AR was used. C, heat-induced AR
with citrate was used and staining of the muscle layers (m) is intense. Background is minimal. Submucosa(s); muscularis mucosa
(arrowhead). All sections were stained by LSAB+ peroxidase-3,39-diaminobenzidine (DAB) for myosin smooth muscle. Bar 5 130 mm.
Figure 5. Labeled streptavidin–biotin peroxidase method. This 3-step method consists of primary antibody-binding tissue
antigens, a secondary biotinylated antibody recognizing the primary antibody, and avidin–peroxidase complexes that will bind the
secondary antibody via avidin–biotin bonds (reprinted from Ramos-Vara43
with permission from Allen Press).
Figure 6. Polymer-based technology. This is a 2-step method. The primary antibody will bind tissue antigens. The
immunoglobulin–peroxidase–polymer complex will bind via immunoglobulins to the primary antibody. This method does not involve
avidin and biotin molecules (reprinted from Ramos-Vara43
with permission from Allen Press).
398 Ramos-Vara et al.

9. is that the boiling temperature is not affected by the
atmospheric pressure, which varies depending on the
altitude.
The relationship between temperature and expo-
sure time is inverse: the higher the temperature, the
shorter the time needed to achieve beneficial results.
The pH of the retrieval solution is important.62
Some
antibodies bind well regardless of retrieval solution
pH, whereas others bind weakly at neutral pH, but
strongly at very low or high pH. Common buffers
used in heat-induced AR are citrate, Tris-HCl, and
EDTA (ethylenediamine tetra-acetic acid). A low pH
buffer (acetate, pH 1.0–2.0) appears especially useful
for nuclear antigens. The significance of the chemical
characteristics of the retrieval solution, particularly
the buffer, is unclear.
Enzymatic digestion. Enzymes are thought to
digest the tissue to some degree, allowing antibodies
to recognize antigenic sites. This digestion is
nonspecific and some antigens might be negatively
affected by this treatment.68
Commonly used enzymes
include trypsin, proteinase K, pepsin, pronase E, and
ficin. A combination of heat and protein digestion
may be necessary to demonstrate some antigens.
Detergent-induced antigen retrieval. Detergents
solubilize membrane proteins by mimicking the lipid-
bilayer environment.54
They form mixed micelles that
consist of lipids and detergents as well as detergent
micelles that contain proteins (also known as
surfactants), thereby decreasing the surface tension of
water. They are usually incorporated into the dilution/
rinse buffers. Examples of detergents include ionic
detergents, bile acid salts, and nonionic and
zwitterionic types (e.g., Triton R-X 100, BRIJR,
Tween 20, saponin).
Practical approach to standardization of a new antibody
Dilutions of primary antibody. Antibody dilutions
for one type of tissue may not work with different
tissues for the same antigen (e.g., brain vs. lymphoid
tissue). Differences in processing techniques and other
variables may require antibody titration for each
tissue type. Often the manufacturing company has
data on antibody performance in FFPE tissue that
can be used as a guideline for the starting dilution.
Typically 2- to 10-fold dilutions above and below the
manufacturer’s recommended dilution provide a good
starting point. Depending on the antibody type
(monoclonal or polyclonal) and concentration, a
range of 1–5 mg/ml should be used for an initial
titration. The optimal working concentration for
most antibodies is generally from below 1 mg/ml to
3 mg/ml, depending on the detection system utilized.
If an antibody has not yet been tested, a systematic
approach using a wide range of dilutions might be
needed, as well as prolonged (e.g., overnight)
incubations with the primary antibody. Secondary
antibodies (linker reagents) and detection complexes
in commercially available detection kits do not need
to be titrated. For practical purposes, when testing a
new antibody, it is recommended to use 3 sets of 5
slides (15 slides total): one set without AR, one set
with enzymatic digestion, and one set with heat-
induced AR. The first 4 slides in each set will have 2-
fold dilutions of the primary antibody and the fifth
slide should be incubated with the negative reagent
control.44,54
A similar approach has been suggested by
the College of American Pathologists.28
Incubation conditions. The conditions for incu-
bation of the primary antibody depend on the
antibody characteristics (e.g., affinity), environmental
factors (e.g., temperature), section characteristics, and
procedure. Incubation for most routine IHC protocols
is 30–90 min at room temperature. During incubation,
the sections must be completely covered by an adequate
volume of the antibody solution to prevent section
drying and assure even exposure of the tissue to the
antibody solution. Shortened incubation times at 37uC,
longer incubation times at room temperature, or
extended overnight incubation in a humidity chamber
at 4uC is occasionally utilized to achieve adequate
immunostaining.17
Additional tissue treatments (e.g.,
decalcification), length of fixation, and AR technique
can affect incubation of the primary antibody.
Decalcification with weak acids does not seem to
interfere significantly with immunostaining of most
antigens, provided the tissues were previously well fixed
in formaldehyde. Strong acids may affect the detection
of some antigens at various degrees.2,4,35
It is
recommended to use weak acidic decalcifying
solutions (e.g., formic acid) diluted in formalin.
a) Primary antibody characteristics.—There are
many characteristics of the antibody that affect
incubation time. The specificity of the antibody is
important in determining the length of incubation
time to get sufficient binding. Lower-affinity anti-
bodies require longer incubation times (and/or higher
concentrations). Affinity rarely has to do with
whether the antibody is monoclonal or polyclonal.
Other antibody-dependent factors include immuno-
globulin isotype, manufacturer, clone, and lot differ-
ences. Different lots of the same antibody may vary in
concentration or other solution characteristics and
thus require re-evaluation.
b) Environmental factors.—Incubation tempera-
ture is a major determinant of incubation time. In
general, as temperature increases, incubation time
decreases. Some antibodies might be temperature-
sensitive with decreased immunoreactivity at higher
temperatures. Sections with longer incubation times
Immunohistochemical techniques in veterinary diagnostic laboratories 399

10. may dry out if there is inadequate humidity in the
incubation chamber. Performing the incubation in
humidity chambers helps to alleviate this complication.
c) Tissues from different animal species.—Species
of origin of the tissue can dramatically affect
reactivity. Interspecies variations in antibody reac-
tions results from subtle changes in the amino-acid
sequence of a given antigen in a particular species.
Even in the event of interspecies cross-reactivity, the
antibody affinity may be decreased. Prolonged
incubation times, varied AR methods, and/or in-
creased antibody concentration may be needed to
obtain optimal staining. Specificity of the antibody
must be verified for every species tested.
Preservation procedure. The type of tissue
preservation will likely affect the performance of the
primary antibody. Frozen sections typically require
less incubation time than do FFPE tissue sections.
Alcohol or acetone fixation has variable effects on the
incubation characteristics of the primary antibody.
There are commercially available fixatives that
reportedly preserve epitopes better for IHC
techniques than the standard formaldehyde,43
but
these fixatives can be more expensive and less suitable
for routine microscopy.
Choice of detection system. The sensitivity of IHC
tests depends mainly on the detection system used. As
a general rule, the more complex an IHC method, the
more sensitive it is. One step or 2-step IHC procedures
are usually less sensitive than more complex, multistep
procedures. Most detection systems currently used in
diagnostic laboratories are based on a color change
induced by an enzyme attached to the immuno-
complexes bound to a tissue section, after reacting
with its substrate and chromogen. The 2 most common
detection systems are avidin–biotin and polymer-based
nonavidin–biotin systems.
Avidin–biotin systems. These systems are based on
the strong affinity of avidin for biotin. Avidin–biotin
systems are currently the most commonly used
detection systems. Biotin is usually attached to a
secondary antibody that binds a complex of avidin–
enzyme to produce a colored reaction. These methods
are very sensitive (Fig. 5). Problems with endogenous
biotin background, particularly when using harsh
(heat) AR methods or staining tissues especially rich
in biotin (e.g., liver), may occur; blocking is usually
necessary but is also expensive.
Polymer-based nonavidin–biotin systems. These
systems are usually 2-step procedures (can be
increased to a 3-step procedure if more sensitivity is
necessary; Fig. 6). The first step is the unlabeled
primary antibody; the second step consists of a
polymer containing numerous secondary antibodies
as well as numerous molecules of enzyme.59,75
These
methods are faster than most current avidin–biotin–
based methods, do not produce the background
generated by endogenous biotin, and have
comparable or sometimes superior sensitivity to
avidin–biotin methods. They are also more expensive.
Additional detection systems. There are multiple
variations to the above-mentioned systems aimed at
increasing the sensitivity of the reaction. Examples of
highly sensitive methods are the tyramine–biotin
method (very sensitive but can create high
background sometimes) and immunorolling circle
amplification, which is a nonavidin–biotin method.43,60
The most commonly used enzymes are peroxidase and
alkaline phosphatase.43,54,64
There are no rules of thumb in selecting a detection
system. The choice of system will depend on several
factors: 1) degree of expertise/experience of the
technician; 2) type of antigens to be detected (some
do not need very sensitive methods because they are
abundant); 3) number of tests (antibodies) available
(different antibodies may require different detection
systems); 4) species idiosyncrasies (e.g., amount of
endogenous biotin in tissues); 5) budget; and 6) best
signal-to-noise ratio when combined with the AR
method used.
Test validation
Validation of a diagnostic test is, in general, the
process of optimizing the test method (reagents and
protocols) and determining the performance charac-
teristics of the test (Fig. 7). Requirements for test
validation are published for AAVLD accreditation
and provide useful general scientific definitions and
practical steps for classifying laboratory tests as
‘‘validated for use,’’ but are not specific for IHC
assays (Essential Requirements for an Accredited
Veterinary Medical Laboratory Version 4.1, Section
5.4.3, 2006. Published by AAVLD). For most IHC
assays, this involves detecting any cross-reactivity of
the selected antibody with unrelated antigens, and
cross-reactivity among different tissues and among
different species. Moreover, validation examines the
variables that affect the IHC reaction, such as fixation
time and storage of paraffin blocks or storage of
unstained tissue sections. Last, but not least, valida-
tion of an IHC test may include comparison of results
among different laboratories using similar tech-
niques.9
When possible, validation compares the
sensitivity of IHC detection to the gold standard
method of detection for the antigen in question. It
may also compare staining patterns and sensitivity
with other antibodies targeting the same antigen.
Validation is usually an ongoing process and gener-
ally requires extensive testing. Although the AAVLD
IHC subcommittee believes that minimum standards
400 Ramos-Vara et al.

11. for validation are needed, the exact validation
procedure will depend on the selected antibody and
the laboratory (in-house validation). As with stan-
dardization, one goal of validation is to produce the
same result regardless of the IHC method used or the
laboratory where the test is performed. The primary
aim among laboratories should be documentation of
their validation procedure(s) as it applies to their
methods for the IHC detection of an antigen.
Cross-reactivity
Two main types of cross-reaction are considered:
antigen cross-reactivity and species cross-reactivity. It
is assumed that testing conditions (e.g., fixation,
tissue processing, antigen retrieval, incubation times,
detection kits) are the same as those used during
standardization of the IHC test.
Antigen cross-reactivity. The type of antigen (i.e.,
structure, amino-acid sequence) affects cross-
reactivity. A literature search may reveal reports of
antigen cross-reactivity. Lack of cross-reactivity in 1
species does not preclude this potential problem in
other species.
a) Infectious diseases.—For antibody-targeting bac-
teria, protozoa, and fungi, it is suggested that the
cross-reactivity of an antibody to other bacteria/
protozoa/fungi that are morphologically similar,
belong to the same group, or produce similar lesions
in the organ of interest, be determined. For example,
an antibody to Leishmania sp. should be tested against
other protozoa (e.g., Trypanosoma, Eimeria, Isospora,
Cryptosporidium, Giardia, Neospora, Toxoplasma, Sar-
cocystis; Fig. 8) and morphologically similar fungi
(e.g., Pneumocystis, Histoplasma, Sporothrix). For
antibody-targeting viruses, it is necessary to test against
other viruses within the same group that affect the
same tissue or produce similar lesions. For example, an
antibody to Canine parvovirus should be tested in
tissues infected with Canine coronavirus, Canine
distemper virus, and Canid herpesvirus.
Some lack of specificity does not necessarily
preclude the use of an antibody provided it is
documented and can be interpreted. An example of
this would be the use of an antiserum raised against
Measles virus nucleoprotein, which reacts with most
known morbilliviruses, for the detection of Canine
distemper virus in dogs. Some mAb against infectious
agents will cross-react with cell organelles (Fig. 9).
b) Neoplastic diseases.—Developing IHC protocols
for antibodies to specific cell types or cell components
requires one or more of the following comparisons:
i. Comparison of immunoreactivity of primary
tumors and their metastases.45,48
Figure 7. Validation of an immunohistochemical test. Factors to be included in the immunohistochemistry validation are antigen
cross-reactivity, species cross-reactivity, fixation, equipment used, storage of reagents, and test results.
Immunohistochemical techniques in veterinary diagnostic laboratories 401

12. ii. Determination of immunoreactivity in other
neoplasms that would be included in the differ-
ential diagnosis (e.g., oral spindle cell sarco-
mas).46,48,50,53
iii. Comparison of immunoreactivity between non-
neoplastic and neoplastic tissues (antigen expres-
sion may be lost or expressed de novo in the
neoplastic phenotype).51
iv. Determination of differences in immunoreactiv-
ity in tumors with variable cell phenotypes (e.g.,
well-differentiated and poorly differentiated) in
the same tissue or different tissues (e.g., mela-
noma).45,48
v. Determination of cross-reactivity of an antibody
targeting a particular cell type in metastatic
tumors that can be present in the same organ
(e.g., hepatocellular tumors and tumors metasta-
sizing to the liver).49
vi. Determination of differences in reactivity be-
tween native and mutated antigens (e.g., p 53).
vii. Determination of prevalence of immunoreactiv-
ity of various antibodies in a variety of tumors or
tissues to determine the relative utility of a
particular antibody for supporting the diagnosis
of a cell type based on the IHC reaction.51,53
Species cross-reactivity. This problem is unique to
veterinary medicine. Although some antigens can be
detected under similar conditions in different species,
it should be assumed that antigen detection will vary
among species.52
Identical antibody clones that target
the same antigen can differ in reactivity among
species (Fig. 10). Detecting an antigen in a new
species may be difficult and laborious. Nevertheless, it
is advisable to restandardize (optimize) a test for each
species, including incubation times, concentration of
the primary antibody, and AR. A literature search or
consultation with the antibody manufacturer and
extensive testing of similar cases in a given species are
recommended to determine species cross-reactivity.
For diagnostic purposes, an IHC report should state
the degree of confidence in the results based on the
laboratory’s or others’ experience (including pub-
lished material). The use of tissue arrays from
different animal species permits rapid screening for
interspecies cross-reactivity.31,39,40,43
Other advantages
of the tissue microarray are savings in reagents and
reduction in results variability; disadvantages include
a) selection of the sample is critical because of its
small size, and b) highly qualified personnel are
needed to prepare the array.
Effects of fixation, postfixation treatments, and equipment
on immunoreactivity
Different fixatives have different effects on immuno-
reactivity. For each fixative, full standardization of a
test must be done, including incubation conditions,
dilution of the primary antibody, and AR method
(see ‘‘Standardization’’ section). Standardization is
usually done on tissues with known fixation time
(e.g., 1–2 days).
Fixation kinetics studies are recommended for each
antigen or epitope. A positive tissue control should be
fixed for different durations (e.g., 1, 2, 4, 7, 10, 14, 21,
Figure 8. Antibody cross-reactivity. A, kidney from a horse infected with Bartonella henselae and stained with antibody to B.
henselae. Strong staining in areas containing microorganisms. B, the same tissue was stained with an antibody to Borrelia sp. The
staining is similar in intensity and anatomic location to that seen in panel A. A polymerase chain reaction of the affected tissue using
primers specific for Bartonella sp. and Borrelia sp. demonstrated genome amplification of only Bartonella. This is an example of the
cross-reactivity of some antibodies and the necessity for extensive validation studies to determine the degree of confidence of each
antibody. Glomerulus (g). Immunoperoxidase-3-amino-9-ethylcarbazole (AEC). Bar 5 35 mm.
Figure 9. Antibody cross-reactivity. In this case, an antibody recognizing rotavirus (arrowheads) in a porcine intestine also cross-
reacts with a supranuclear structure (interpreted as the Golgi apparatus) in most enterocytes (arrows). The authors have observed similar
nonspecific reaction with other monoclonal antibodies to Bovine viral diarrhea virus, Bovine coronavirus, and Bovine herpesvirus 1.
Immunoperoxidase-3,39-diaminobenzidine (DAB). Bar 5 17 mm.
402 Ramos-Vara et al.

13. 20 days) and tested under identical conditions to
determine the effects of fixation time on reactivity
(i.e., intensity and number of cells or organisms
detected; Fig. 11).45,48
Automated stainers are designed to duplicate
manual staining procedures and can be used to ensure
uniform application of all steps of the process. Thus,
the use of automated equipment offers a uniform and
standardized microenvironment for testing, which will,
in turn, result in intralaboratory run-to-run consist-
ency.64,77
When results among different laboratories
differ considerably, these technical differences should
be considered as a possible cause.
Storage of paraffin blocks and tissue sections
It is the authors’ and others’ opinion that paraffin
blocks remain stable for years in terms of antigenicity.
However, only controlled studies on the effect of
nucleic acid detection on archival paraffin blocks
have been published.25
Any deleterious effects of
prolonged storage on tissue antigenicity in paraffin
blocks, if they happen, may differ among antigens.
For practical purposes, consider this factor if
inconsistent IHC results occur when using old
paraffin blocks.
Storage of unstained paraffin control tissue sec-
tions increases efficiency but may adversely affect
immunoreactivity (tissue section aging), depending on
the antigen of interest.24
Photo-oxidation of tissue
sections is involved in loss of antigenicity.13
Tissue
section ageing is a rather common problem with
nuclear antigens (e.g., Ki67, estrogen receptor,
p53).11,19,25,30,34,36,38,42,54,70,72
When there is decreased
intensity or loss of reaction in stored control tissue
sections, repeat the test with another stored control
tissue slide and a freshly cut control tissue section. If
the change is limited to stored sections, discard any
remaining unstained control slides.
What constitutes a positive/significant result?
There is no single answer to this question. Immu-
nohistochemical assays detect analytes (infectious
agent or tumor marker antigens) in tissues but do
not necessarily diagnose disease, which may require
interpretation of IHC results in correlation with other
clinical or laboratory findings. For an infectious agent,
detecting one organism indicates infection, but it is
another matter to prove that the agent caused disease.
For neoplasms, this answer is even more difficult. In
the literature, the percentage of positive cells required
to confirm tissue origin of a tumor varies considerably,
and often this striking variation is reported for
detection of the same antigen by different authors.
Some pathologists prefer the words ‘‘detected’’ or ‘‘not
detected’’ rather than ‘‘positive’’ or ‘‘negative.’’ This
seems semantic but underscores not only the need to
interpret subjectively a colored reaction, but to do so in
the context of the disease.61
Controls
Positive tissue control
Positive tissue control is defined as tissue that is
known to contain the antigen of interest detected by
identical IHC methods to those used in diagnostic
cases. Fresh-fixed surgical specimens (or biopsy
tissue) are preferred over necropsy material whenever
possible, since autolysis may affect staining results.
Positive tissue controls must be fixed and stained in
the same way as the diagnostic case tissue for every
antibody and procedure used.55,64
The presence of the
antigen in the tissue control should be confirmed by
another method (e.g., PCR, virus isolation). For most
laboratories, control tissues are generally obtained
‘‘in-house’’ because variables such as fixation and
processing are the same as those used with diagnostic
case material. Control tissues obtained from another
laboratory or from a commercial source may have
been fixed/processed in a different way than the test
tissue. However, the use of in-house standards does
not ensure reproducibility of a particular IHC assay
between different laboratories.
Multi-tissue blocks and cell lines. Various methods
for the simultaneous study of multiple FFPE tissues
in a single histologic section have been reported. The
‘‘sausage technique’’ has been used heavily in the
past.10
In this method, long thin strips of fixed tissues
are drawn into a tube of unfixed small intestine. The
entire sample is then fixed, resulting in a tight column
that can be cut into blocks and embedded. Paraffin-
embedded tissue microarrays have been proposed as a
reference control standard for human IHC laborato-
ries, primarily for use in tumor diagnosis.29,39,40,43
It is
recommended that validation studies be carried out
on multi-tissue control blocks containing both
known-positive and known-negative normal and
tumor tissues.64
In veterinary medicine, species-
specific tissue microarrays are not commercially
available. Species-specific tumor cell lines are being
used for IHC controls (e.g., IHC for HER-2/neu).55,57
This approach provides an identical tissue control
among different laboratories and is an excellent way
to compare results among them.
Reference control tissues for infectious diseases. The
presence or absence of a particular infectious agent in a
tissue should be assessed by previously published
IHC reactivity patterns (if available) plus another
non-IHC diagnostic method.8
For example, Listeria
monocytogenes–control tissue could be assessed
by IHC (immunoreactivity of extracellular or
Immunohistochemical techniques in veterinary diagnostic laboratories 403

14. Figure 10. Species reactivity differences. Eyelid, horse. This tissue was incubated with two different antibodies to vimentin, a
mouse monoclonal antibody (A) and a rabbit monoclonal antibody (B). Reactivity is only observed in the tissue incubated with the
mouse monoclonal antibody. There is spurious staining in the mucosal epithelium in panel B (asterisk). The rabbit monoclonal antibody
to vimentin reacts as expected in other animal species (e.g., dog, cat). Immunoperoxidase-3,39-diaminobenzidine (DAB). Bar 5 65 mm.
Figure 11. Effects of prolonged fixation. A, tissue was fixed for 24 hr. B, tissue was fixed for 11 weeks. Bartonella organisms
(asterisks). Note that there is no staining in tissue fixed for several weeks. Sensitivity to fixation is variable for each antibody; thus, the
need to evaluate fixation kinetics in any new immunohistochemical procedure, if possible. Immunoperoxidase-3,39-diaminobenzidine
(DAB) for Bartonella. Bar 5 33 mm.
404 Ramos-Vara et al.

15. phagocytized bacilli) and by bacterial culture.
Similarly, Bovine viral diarrhea virus (BVDV)–control
tissue could be assessed by IHC (immunoreactivity in
cytoplasm of infected cells) and by virus isolation or
PCR. In other instances, the presence of characteristic
lesions for a particular disease (e.g., intranuclear
inclusions observed with hematoxylin and eosin [HE]
staining) can be used as a standard reference control.
Immunohistochemical protocols for infectious dis-
eases should, whenever possible, also be species-
matched with test specimens; alternatively, tissues
from other species affected with the same infectious
agent can be used (e.g., BVDV identified in diagnostic
case material from cervids using a bovine control
tissue). Nonspecific binding of primary antibody
(species-specific molecular mimicry) or secondary
(linker) reagents may occur among different species.
A good example is the use of goat polyclonal
antiserum specific for Neospora caninum on goat
tissue. The anti-goat IgG secondary reagent used in
the IHC assay can bind excessively to endogenous
goat IgG in the tissue resulting in such severe
background ‘‘noise’’ that visualization of individual
immunoreactive tachyzoites is impossible.
Reference controls for neoplastic diseases. Many
times, the only way to verify the presence of an
antigen is by morphology, which is not always an
accurate predictor for the presence or absence of a
particular antigen. Furthermore, a negative result for
tumor markers does not necessarily rule out a
particular neoplasm because the cell marker of
interest may not be expressed in a very poorly
differentiated cell population. Ideally, controls for
tumor cell markers should consist of the neoplastic
tissue of interest, which expresses different intensities
of reactivity.64
Control tissues for IHC tumor markers
should also be species-matched with test specimens
because the detection of an antigen in a particular
tumor and animal species does not guarantee similar
results in a different animal species.
Negative tissue control
Negative tissue control is defined as tissue that is
known not to contain the antigen of interest.64
At
least 1 ancillary test (e.g., PCR, virus isolation)
performed on the tissues/organ systems of the same
animal should be used to rule out the presence of the
antigen of interest. Both positive and negative in-
house tissue controls should be used in each test and
should be processed in the same manner as the case
material. Similarly, when using multitissue blocks for
antibody validation studies or as tissue controls, the
specimens must be fixed and processed in the same
manner as the case material.64
When dealing with
cellular antigens (e.g., cytokeratin, vimentin), positive
control tissue should have areas expressing variable
amounts of the specific antigen. Weakly stained areas
can be used to detect subtle changes in antibody
sensitivity.64
In practice, only 1 tissue control is used
because of the common presence of both positive and
negative cells within the control.
Internal positive tissue controls
Internal positive tissue controls are present in
diagnostic case tissues. An example is the detection
of smooth muscle markers or vimentin in normal
blood vessels. The presence of positive staining in
these areas indicates appropriate immunoreactivity.
With this type of control, there is no fixation variable
between the control tissue and the diagnostic case
tissue.14
Although some vimentin antibodies have
been used to demonstrate overfixation effects due to
their sensitivity to fixation,55
this type of test may not
be relevant with the current AR methods.
Reagent (antibody) controls
Negative reagent controls are used to confirm the
specificity of the test and to assess the degree of
nonspecific background staining present by omitting
the primary antibody. Commonly, the primary
antibody is replaced by 1 of the following methods:
1) antibody diluent, 2) same species nonimmune
immunoglobulin of the same dilution and immuno-
globulin concentration, 3) an irrelevant antibody, or
4) buffer.55,64
These methods will assess the degree of
cross-reactivity of the primary antibody, and the
degree of nonspecific binding by the labeling (sec-
ondary) antibody and detection system; only methods
2 and 3 approach the true significance of negative
controls. There are also commercially available ready-
r
Figure 12. Nonspecific staining. Knowledge of the expected antigen distribution of the reaction within a cell or tissue is mandatory
before using a test for diagnostic purposes. In this case, an antibody to CD79a stained only the nucleus of plasmacytoma cells. This
reaction is not considered specific based on the current knowledge of the location of this antigen (cytoplasmic and cytoplasmic
membrane). Bar 5 60 mm. The lower inset is a detail of the aberrant staining; the upper inset depicts a typical cytoplasmic staining of
CD79a in a plasmacytoma. Immunoperoxidase-3,39-diaminobenzidine (DAB). Both inset bars 5 5 mm.
Figure 13. Equipment maintenance. One of the possible causes of unusual staining in immunohistochemistry is inadequate
maintenance of equipment. The heat unit (arrows) of the steamer needs to be free of salts that will progressively deposit when using tap
water. A, clean unit. B, unit with severe salt deposition. The heat unit in panel B could not reach the expected temperature.
Immunohistochemical techniques in veterinary diagnostic laboratories 405

16. to-use universal negative reagents for rabbit and
mouse primary antibodies.
If the diagnostic workup requires a panel of
antibodies each of the same isotype and similar
concentration and derived from the same species, the
panel of antibodies itself may serve as a set of
irrelevant reagent controls; thus, the need for multiple
negative controls is eliminated. If this method is
utilized, separate controls should still be run for each
different type of protocol (e.g., for each protocol with
a difference in the AR method or difference in the
detection system).64
Storage and handling of reagents
The shelf life of many reagents is directly linked to
appropriate storage. The shelf life of many primary
antibodies beyond that indicated by the manufacturer
can be significantly extended with proper handling and
storage.6,67
However, use of a reagent beyond the
manufacturer’s expiration date is not covered by its
warranty and has to be properly documented. General
considerations for storage of reagents include:
1. Storage containers must be made of nonreactive
material that does not alter the reagent by
adsorption or polymerization of components
within the reagent or addition of components to
the reagent.
a. Plastics: Polypropylene and polycarbonate
are nonreactive and are routinely used for
storage of primary antibodies at 220uC or
270uC. Containers that can be tightly sealed
are necessary to prevent desiccation.18
b. Borosilicate glass: This material is used when
storing reactive reagents. When storing di-
luted antibody preparations, addition of
0.1–1.0% bovine serum albumin (BSA) may
be necessary to decrease polymerization and
adsorption in any container used.26
2. Primary antibodies should be stored frozen as
concentrates in appropriately sized aliquots in
keeping with manufacturer’s recommendations.
Freezing minimizes denaturation of proteins and
reduces excessive mechanical action or contact
with air.26
Once thawed and diluted, the antibody
preparation should be stored at 2–8uC. Repeated
freezing and thawing of the concentrated anti-
body can result in significant loss of reactivity.
3. The majority of buffers are stored at 2–8uC to
prevent microbial growth.
a. Sodium azide (NaN3) is often used in
commercial concentrated primary antibody
preparations as a preservative. For in-house
antibodies, use 0.02% NaN3. To avoid
enzyme inhibition by NaN3, use 0.01%
merthiolate in conjugates.26,66
Do not use
preservatives in buffers or antibody diluents.
b. Close inspection of buffers and other re-
agents for signs of microbial contamination
or precipitation of salts is necessary each time
the reagent is used.
4. Proper labeling of stored reagents is essential.
Each reagent label should include the name of
reagent, date made or opened, expiration date,
and name or initials of individual who made or
opened the reagent.
5. Stock solutions often have a longer storage life
compared to working solutions. Working solu-
tions can be prepared daily for immediate use or
stored for short term.
6. Substrates (or chromogens) should be freshly
prepared for each run or as directed by the
manufacturer.
7. Storage at 270uC does not provide significantly
longer shelf life than storage at 220uC. Storage of
reagents in frost-free freezers is not recommended
because of their programmed fluctuations in
temperature that contribute to increased polymer-
ization and denaturation of many proteins.
8. Routine temperature checks of all storage areas
and appropriate relevant documentation are
recommended.
Quality assurance/quality control and interpretation of
IHC results
Quality reflects on the procedural and technical
aspects of the IHC test and is aimed to reduce and
correct deficiencies in an analytical process. Each step
of an IHC test is defined by quality control (QC)/
quality assurance (QA) standards that have been
previously addressed. Quality control issues, as they
pertain to daily records and the standardization of
new primary and secondary antibodies and controls,
are discussed here.
Daily quality assurance/quality control
Each laboratory should establish standard opera-
tion procedures (SOPs) for their routine histology
laboratory. This would include daily, weekly, and
monthly schedules for cleaning, maintenance, and
monitoring logs, along with a daily check of
equipment such as oven temperature for drying slides,
temperature of water baths, pH meter, etc. Buffers
should be made using distilled deionized water
(ddH2O) and their pH checked and adjusted, if
necessary, before use. When enzymes are used for
AR, they should be prepared shortly before use.
There are also ready-to-use commercial enzyme
406 Ramos-Vara et al.

17. solutions for AR. Note the storage conditions and
expiration dates for any reagent.
A worksheet should list the stains requested and
number of slides to be stained. The slides should be
labeled accordingly and double-checked against the
IHC request forms submitted by the pathologist.
Fresh working solutions of primary antibody
should be prepared according to their storage
requirements. The expiration dates of the antibody
and the working dilution should be indicated on the
tube. Xylene/xylene substitute and graded alcohols
used for deparaffinization, rehydration, and dehydra-
tion should be changed before they lose significant
strength. It is recommended to use AR solutions only
once; however, each laboratory should establish their
own protocol for reusing AR solutions.
When using an automatic stainer, a ‘‘Before
Staining Checklist’’ should be observed.
The following are some important items to include
on the list:
N Check to ensure there is room for waste; empty
the waste containers if necessary.
N Check quantities of reagents for the run: ddH2O,
buffers, IHC kit reagents, and chromogen.
N Check to ensure correct programming is being
used for the run.
N Check slide labels as the stainer is loaded.
N Check the quantity and placement of the IHC
reagents.
N Remove all caps from reagent tubes.
The maintenance schedule for the automated stainer
should be followed and documented.
Primary antibodies. The date received, date
aliquoted, date of dilution, and expiration date must
be written on the appropriate containers. New lots of
antibody should be tested and titrated before use. It is
best to prepare only the quantity of reagents required
in a given run. Aliquots should be made of stock
primary antibody in volumes convenient for
preparation of working dilutions.
Secondary reagents. Most reagents are commer-
cially prepared and standardized by the manufacturer.
Reagents should be stored per manufacturer’s recom-
mendations. Batch numbers should be recorded. Keep
in mind that guarantee of a reagent only applies when
the product is used as specified by the manufacturer.
Control tissues and stains. See ‘‘Controls’’ section.
Internal quality assurance/quality control
A bi-annual internal QA/QC check for the quality
of IHC slide interpretation is recommended. In large
diagnostic laboratories, randomly selected slides
should be reviewed by other pathologists on a
rotational basis. The slides should be interpreted by
the pathologist and a written evaluation of the quality
of the previous interpretation should be provided.
Written comments on the technical aspects and the
overall quality of the slides should be made.
External quality assurance/quality control
The goal for interlaboratory comparisons is to
document variations in reactivity (e.g., positive vs.
negative, intensity, number of positive cells) among
different laboratories and to set minimum QA/QC
standards to create optimal protocols that could be
shared by all laboratories.74
Interlaboratory stan-
dardization is difficult. Even in human medicine, only
a handful of IHC tests (e.g., Hercep test) have been
validated in such a way that different laboratories
could perform tests with consistent results, and not
without controversy.12,27,58
Veterinary diagnosticians
are still a long way from achieving interlaboratory
standardization. As a previous analysis pointed out,
‘‘Whether we like it or not, the practice of anatomic
pathology is to some extent subjective, although we
all strive for as much objectivity and reproducibility
as possible in our daily work.’’23
There are 2 main approaches to interlaboratory
standardization:
1. Technical equivalency testing of an IHC test.
Unstained slides are distributed among different
laboratories for performance of the requested
immunostains; results are compared among dif-
ferent laboratories or with those of the reference
laboratory. This test determines the quality and
interlaboratory consistency of the IHC testing.
2. Diagnostic equivalency testing using IHC testing as
an adjunct. In this case, both the pathologist’s
proficiency and the quality of IHC testing are
evaluated. A brief history and description of the
tissue examined is included with tissue sections. The
pathologist must make a diagnosis based on routine
(e.g., HE) staining and support it with appropriate
IHC testing. Results could be reviewed by an
established committee that would address possible
differences among participating laboratories.73
The current working proposal of the Pathology
Committee for the AAVLD Program for Interlabora-
tory Comparison of Infectious Agent Immunohisto-
chemical Assays recommends having a reference
laboratory (proposed to be the National Veterinary
Laboratory Services, Pathobiology Section, Ames,
IA) send participating laboratories a set of unstained
FFPE sections for immunostaining and interpreta-
tion. Results should be qualitative (detected/positive,
not detected/negative) and not quantitative. Partici-
pating laboratories would return the slides and
interpretations to the reference laboratory for evalu-
Immunohistochemical techniques in veterinary diagnostic laboratories 407

18. ation and feedback. For cellular antigens, a similar
approach may be considered in the future.
Immunohistochemical report and interpretation
If not included in the standard report format, the
IHC report should contain demographic information
pertinent to the case, the tissue that was tested, and
the antibody used, or the antigen in question should
be listed. Other details of the IHC test should be filed
in the laboratory. Results for infectious antigens have
to be reported as ‘‘detected’’ or ‘‘not detected.’’ In
addition, the interpretation for infectious disease
IHC/immunocytochemistry (ICC) tests should in-
clude a disclaimer that any lack of detectable antigen
may be a result of the absence of the antigen in this
particular section and/or caused by technical aspects,
such as prolonged fixation. In the case of tumor
markers, it may be important to include a description
of the cellular location (e.g., cytoplasmic, membrane,
nuclear) and intensity of staining and percentage of
positive cells, along with an interpretation of the
significance of the test results (Fig. 12). When the
antigen location is atypical, the report should
mention this. Personnel who read IHC slides should
be familiar with the antibodies used and their
specificity and sensitivity. The authors highly recom-
mend having a trained veterinary pathologist for QA/
QC purposes review daily IHC staining.
Troubleshooting guide
Weak or no staining of positive control and
weak or no staining of test slides
Consider the following:
1. Problem: all slides from some primary antibodies
in the run are affected. Action: check for
inadequacy of the primary antibody, method
incompatibility, primary and link antibody in-
compatibility, or inadequate AR (Fig. 13).
2. Problem: the whole run is affected. Action: check
assay log and adequacy of reagent volumes and
sequence of reagent delivery to the slide. Deter-
mine if reagents were delivered to all slides (e.g.,
buffer, chromogen).
3. Problem: if it is hit-and-miss throughout the run,
consider technical problems or problems with the
tissues. Action: check for inadequate sequence of
reagents, unbalanced autostainer, and inadequate
drop zone.
Checklist assuming that the positive control tissue
has been proven:
1. Review assay logs/datasheets/checklists to be sure
that no step was omitted and that all steps were
done in correct sequence.
2. Review assay logs/datasheets/checklists for ap-
propriate incubation times and temperatures.
3. Check protocol for appropriate pretreatment or
AR.
4. Make sure the linking antibody is compatible
with the primary antibody.
5. Check for out-of-date reagents or antibodies and
ensure that they have been properly stored
according to package inserts.
6. Ensure that the primary antibody was properly
diluted.
7. If a reagent was stored frozen, determine that it
was not repeatedly frozen and thawed.
8. Check deparaffinization protocols and reagent
quality (e.g., not overused).
9. Ensure that the substrate-chromogen was freshly
and properly prepared.
10. Determine that the slides did not dry out at any
time during the procedure.
11. Check the pH of the buffer solutions.
12. Other less common but possible problems:
a. Sodium azide in the wash buffers may inhibit
peroxidase reactions.
b. Certain commercial phosphate buffer solutions
may inhibit alkaline phosphatase activity.
c. Use of inappropriate counterstain or dehydra-
tion steps with alcohol-soluble chromogens.
d. Exposure of tissues to temperatures of .60uC
during embedding or drying.
e. Leaving too much buffer on slides after
washes. This dilutes reagents.
f. Excessive counterstaining interfering with
interpretation.
g. Prolonged storage of tissue sections.
13. Try a new positive control tissue.
Inadequate or no staining of test slide and
adequate staining of positive control slide
1. Problem: this is a true negative result; the antigen
in question is not present in the test tissue.
Action: none.
2. Problem: the antigen is present in the test tissue,
but in a concentration below the method’s
detection limits. Action: consider using an ampli-
fication procedure, or increasing the primary
antibody concentration, incubation time, or
temperature or a combination thereof.
3. Problem: the test tissue is over- or underfixed.
Action: modify AR protocol.
4. Problem: the test tissue is from a different species
than the control tissue and has different reactiv-
ity with the primary antibody. Action: validate
408 Ramos-Vara et al.

19. the IHC test with same species control and test
tissues.
5. The following are other potential but uncommon
problems:
a. Excessively high (.60uC) embedding or
drying temperature was applied to the test
slide only (assuming the positive control
tissue and the test tissue are on different
slides). Action: check oven temperature.
b. Prozone effect occurred due to high concen-
tration of the primary antibody in the test
tissue. Action: retitrate the primary antibody
for the test slide.
c. A technical error (e.g., inadequate determi-
nation of the drop zone by the autostainer;
unbalanced platform of autostainer or slide
rack(s); insufficient amount of reagents) has
occurred. This may occur on the test slide but
not the positive control slide (assuming the
positive control tissue and the test tissue are
on different slides) or the test tissue but not
the positive control tissue (assuming the test
tissue and positive control tissue are on the
same slide). Action: check autostainer balance
status and dropping zone, respectively.
No staining of positive control slide and
adequate staining of test slide
1. Problem: technical error in the staining or
handling of the positive control slide. Action: the
log or checklist should be reviewed; if everything
is in order, the assay should be repeated. For
control blocks for infectious diseases, the antigen
to be tested may not be present through the entire
block.
2. Problem: tissue section aging. Lack of staining in
the tissue section control might be a result of tissue
section aging. Action: compare staining of stored
tissue sections with a fresh cut control section.
Excessive background staining
Prestaining problems.
1. Inadequate fixation, necrosis, and autolysis
2. Tissue sections allowed to dry out. Action: reduce
incubation time; incubate in a humidified chamber.
3. Sections not completely deparaffinized. Action:
use fresh dewaxing solutions.
4. Slide adhesive inappropriate or too thick. Action:
use adhesives specific for IHC or positive-
charged slides.
5. Tissue section too thick. Action: prepare thinner
sections.
6. Inappropriate AR used. Action: re-evaluate AR
conditions.
7. Incubation temperature too high. Action: reduce
temperature.
Blocking problems.
1. Endogenous enzyme activity not suppressed.
Action: increase concentration of blocking agent.
2. Inadequate protein blocking. Action: use a
different blocking agent.
3. Inadequate blocking of endogenous avidin-bind-
ing activity. Action: use an avidin–biotin blocking
step or use a nonavidin–biotin detection method
(Fig. 14).
4. Inadequate blocking of endogenous biotin. Ac-
tion: use an avidin-biotin blocking step or a
nonavidin–biotin detection method.
5. Blocking serum from improper species. Action:
use blocking serum from same species as the link
(secondary) antibody.
Primary antibody problems.
1. Primary antibody too concentrated. Action:
retitrate primary antibody (Fig. 15).
2. Primary antibody incubation time too long.
Action: reduce incubation time.
3. Primary antibody is from a similar or identical
species as the test tissue (e.g., mouse on mouse
[MOM], rat on mouse). Action: use specific
protocols (i.e., MOM or similar kits) or addi-
tional blocking steps.
4. Inadequate buffer washes (inappropriate buffer
ion concentration). Action: modify ionic strength
of the buffer solution.
Secondary antibody problems.
1. Secondary antibody and label concentration too
high.
2. Secondary antibody and label incubation time
too long.
3. Buffer washes insufficient.
4. Secondary antibody recognizes endogenous (tis-
sue) immunoglobulins (Fig. 16).
5. Microbial contamination of primary antibody
solution. Action: reformulate from fresh stock.
Add antimicrobial preservatives. Respect proper
storage conditions for antibody solutions.
Chromogen and counterstain problems.
1. Chromogen concentration too high. Action:
reduce concentration of chromogen.
2. Chromogen allowed to react too long. Action:
reduce incubation time with chromogen.
3. Buffer washes insufficient. Action: prolong buffer
washes.
Immunohistochemical techniques in veterinary diagnostic laboratories 409

20. Figure 14. Background caused by endogenous avidin–biotin activity (EABA). Kidney, dog. A, strong background staining
(EABA) on a section incubated with negative reagent control and an avidin–biotin detection system. B, a consecutive tissue section
incubated with a reagent negative control and EnVision+, a nonavidin–biotin detection method. There is no background staining. C,
staining for cytokeratins in renal epithelium. There is no background staining. EnVision+ peroxidase-3,39-diaminobenzidine (DAB). Bar
5 60 mm.
Figure 15. Background staining caused by antibody concentration. Mammary gland, dog. A, the primary antibody to p63 is too
concentrated and immunohistochemical reaction is present not only in the nucleus, but also in the cytoplasm of epithelial and stromal
cells. B, the antibody concentration was optimized and only nuclei display staining (the nucleus is the typical location of this antigen).
Immunoperoxidase-3,39-diaminobenzidine (DAB) for p63. Bar 5 60 mm.
Figure 16. Background staining caused by secondary (link) antibody. This is a common problem when using secondary antibodies
recognizing antibodies from the same species as the tissue examined. In this case, 2 detection systems (LSAB+ for A and B) and
EnVision+ (for C and D) were used. Goat tissue stained for cytokeratins using LSAB+ detection system (the secondary antibody
recognizes endogenous goat immunoglobulins) has a strong nonspecific staining in both the positive (A) and negative (B) reagent
sections. Specific staining for cytokeratins in epithelial cells (arrowheads); nonspecific staining in the stroma (asterisk). Note that the
negative reagent control section (B) does not have staining in epithelial cells. C, when similar tissues were stained with the same antibody
using EnVision+ as detection system (it does not recognize goat immunoglobulins), only specific staining of epithelial cells was observed.
D, as expected, the negative reagent control does not show staining. Immunoperoxidase-3,39-diaminobenzidine (DAB) for cytokeratins.
Bar 5 60 mm.
410 Ramos-Vara et al.

21. 4. Counterstain obscures the IHC reaction. Action:
use a different counterstain that does not
interfere with IHC staining.
An excellent review of troubleshooting is available by
Atwood in Immunohistochemical Staining Methods.5
Conclusion
Immunohistochemistry is a well-established ancil-
lary technique to facilitate the diagnosis of infectious
and neoplastic processes in animals. This article has
reviewed the main factors involved in the IHC test,
from the preservation of samples and preparatory
steps, followed by the immunohistochemical reaction
and visualization of the reaction, to the interpretation
and generation of an IHC report. Each step of the
IHC test may require troubleshooting and a guide for
this is also included. One goal of this article is to
suggest guidelines to help veterinary laboratories
establish standardization and validation procedures
in diagnostic IHC, thus providing a method of quality
control and quality assurance in a very subjective
discipline.
Acknowledgements
The authors are members of the AAVLD Subcommittee
on Standardization of Immunohistochemistry.
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