when tissue antigens and antibodies get along...cross-linking.51,77 formation of cross-links between...
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Article
When Tissue Antigens and AntibodiesGet Along: Revisiting the Technical Aspectsof Immunohistochemistry—The Red,Brown, and Blue Technique
J. A. Ramos-Vara1 and M. A. Miller1
AbstractOnce focused mainly on the characterization of neoplasms, immunohistochemistry (IHC) today is used in the investigation of abroad range of disease processes with applications in diagnosis, prognostication, therapeutic decisions to tailor treatment to anindividual patient, and investigations into the pathogenesis of disease. This review addresses the technical aspects of immuno-histochemistry (and, to a lesser extent, immunocytochemistry) with attention to the antigen-antibody reaction, optimal fixationtechniques, tissue processing considerations, antigen retrieval methods, detection systems, selection and use of an autostainer,standardization and validation of IHC tests, preparation of proper tissue and reagent controls, tissue microarrays and otherhigh-throughput systems, quality assurance/quality control measures, interpretation of the IHC reaction, and reporting of results.It is now more important than ever, with these sophisticated applications, to standardize the entire IHC process from tissuecollection through interpretation and reporting to minimize variability among laboratories and to facilitate quantification andinterlaboratory comparison of IHC results.
Keywordsantibodies, antigen retrieval, fixation, immunohistochemistry, review, standardization, technical aspects, validation
Paul Ehrlich introduced the term antibody (Antikorper) in
189146 and hypothesized that cell surface receptors bind specif-
ically to toxins in a lock-and-key interaction. However, it was
not until 1941 that antigen detection in tissue sections was
reported, marking the birth of immunohistochemistry (IHC).65
Since then, the number of tests and their specificity and
sensitivity have increased to the point that IHC routinely sup-
plements the morphologic approach to pathology.292 Although
a major early application was in the characterization of
neoplasms, IHC currently has broader and more clinically
oriented applications in diagnosis, prognosis, therapeutic deci-
sion making, and pathogenesis.206,372 In essence, IHC fills the
gap between classic histopathology and molecular pathology.
Immunohistochemistry bridges 3 disciplines: immunology,
histology, and chemistry. The fundamental concept is the
demonstration of antigens within tissue sections by means of
specific antibodies. The immunoglobulin molecule has binding
sites for antigens and for other antibodies (Fig. 1). Antigen-
antibody binding is demonstrated with a colored histochemical
reaction visible by light or fluorescent microscopy.284 In other
words, the basic principle of IHC, as with other ‘‘special’’
histochemical methods, is visual localization of cell or tissue
target molecules based on a satisfactory signal-to-noise ratio.371
Although conceptually simple, the methodology of IHC has
become more complex with more stringent requirements for
sensitivity and specificity.237 The initial simple (direct) methods
produced quick results but lacked sensitivity. Currently,
extremely sensitive methods can detect 1 or multiple antigens
simultaneously or can screen hundreds of tissues in the same
section (tissue microarray technology) for the presence of a
particular antigen. The feasibility of detecting multiple antigens
by IHC has improved dramatically with the use of multispectral
analysis.209 Another critical advance in the 1990s was the
introduction of techniques to ‘‘retrieve’’ antigens that had been
altered by fixation, increasing exponentially the number of anti-
gens detectable in routinely fixed tissues.284
In some cases (eg, prion diseases), IHC is considered the gold
standard to which other techniques are compared. In contrast to
many detection techniques, IHC allows colocalization of an anti-
gen with a lesion, thereby dramatically enhancing diagnostic
interpretation and understanding of the pathogenesis.
1 Department of Comparative Pathobiology, College of Veterinary Medicine,
Purdue University, West Lafayette, IN, USA
Corresponding Author:
J. A. Ramos-Vara, Animal Disease Diagnostic Laboratory and Department of
Comparative Pathobiology, Purdue University, 406 South University, West
Lafayette, IN 47907, USA.
Email: [email protected]
Veterinary Pathology2014, Vol 51(1) 42-87ª The Author(s) 2013Reprints and permission:sagepub.com/journalsPermissions.navDOI: 10.1177/0300985813505879vet.sagepub.com
Figure 1. Immunoglobulin structure. The Fc fragment is delimited by the oval dotted line; the F(ab) fragment, by the oval dashed line. The areawithin the square is the variable region of the F(ab) fragment. Figure 2. Effects of autolysis on immunohistochemistry (IHC). Parathyroidadenoma; dog. Diffuse nonspecific background reactivity in autolyzed tissue, including nuclear labeling (with a cytoplasmic marker) in thyroidfollicular cells (upper inset) and parathyroid chief cells (lower inset). Thyroid gland (asterisk). Parathyroid adenoma (solid circle).Immunoperoxidase-DAB for cytokeratins, hematoxylin counterstain. Figure 3. Effects of overfixation. (a, b) IHC for Bartonella sp in equine fetallung. Strong immunoreactivity at 2 days’ fixation (a) with loss of reactivity at 11 weeks’ fixation (b). (c, d) IHC for cytokeratins; skin, dog. Strongimmunoreactivity at 2 days’ fixation (c); weak reactivity at 7 weeks (d). Immunoperoxidase-DAB, hematoxylin counterstain. Figure 4. Effects ofsuboptimal fixation on IHC. B-cell lymphoma; lymph node, dog. Only the outer rim of tissue was adequately fixed (formalin). Proper hematoxylinand eosin (HE) staining (a) and IHC reactivity are limited to the well-fixed portion of the section. Immunoperoxidase-DAB for CD79a. Figure 5.Effects of alcohol fixation on protein structure. Alcohol interacts with hydrophobic moieties, modifying the tertiary structure of the protein.Modified from Ramos-Vara284 with permission from Veterinary Pathology.
Ramos-Vara and Miller 43
Although IHC has become a routine tool for veterinary
diagnostic and research studies, the variable cross-reactivity
of antibodies among different species raises many challenges
for the comparative pathologist.284 This review addresses
technical aspects of IHC—fixation, antigen retrieval,
antigen-antibody reactions, detection methods, controls,
standardization, validation, and quality assurance—as well
as interpretation. Basic aspects of immunocytochemistry will
be addressed briefly.
Overview of the Immunohistochemical Test
The IHC technique is a combination of immunologic and
chemical reactions visualized with a photonic microscope. The
technique can be divided into 3 phases (Table 1). Phase 1 (prea-
nalytical) starts with sample procurement, followed by tissue
fixation, trimming, and embedding, and ending with tissue
sectioning on a microtome. Phase 2 (analytical) starts with depar-
affination of tissue sections; includes preincubation steps (eg,
antigen retrieval, blocking of nonspecific activities), incubation
with the primary antibody, and labeling of the antigen-antibody
reaction; and ends with slide counterstaining and coverslipping.
Phase 3 (postanalytical) includes interpretation of results and
generation of an IHC report, as well as evaluation of the IHC
controls.369
Preanalytical Phase of IHC
Fixation
Fixation of tissues is necessary to (1) adequately preserve cel-
lular components, including soluble and structural proteins; (2)
prevent autolysis and displacement of cell constituents, includ-
ing antigens and enzymes; (3) stabilize cellular materials
against deleterious effects of subsequent procedures; and
(4) facilitate conventional staining and immunostaining.142
No fixative optimally fulfills all these goals.284 Chemical fixa-
tion is generally used for diagnostic IHC; the most common
chemical fixative is formalin (Table 2).
Effects of Delayed Fixation. Prompt transfer of postmortem spe-
cimens into fixative is critical.149 Delayed fixation can
decrease the mitotic index, with up to 40% reduction when
fixation is delayed more than 12 hours;70,82,151 this reduction
in mitotic index can alter tumor grade.356 Likewise, with
biopsy specimens, once the tissue is removed from the living
body, biochemical alterations include adenosine triphosphate
(ATP) depletion and disruption of sodium, potassium, and
calcium gradients.149 Prolonged hypoxia as a result of
delayed fixation can cause cellular swelling, generation of
reactive oxygen species, and activation of various enzymes,
all of which can change the immunoreactivity of the target
antigen, especially for measurement of apoptosis or detection
of the phosphorylation state of signaling pathways.94,149
Delayed fixation can affect the IHC results in various
ways.79,94,149,223,272 A fixation delay of more than 2 hours
can decrease the detection of breast cancer biomarkers such
as estrogen receptor (ER), progesterone receptor (PR), and
HER2,186 although others have not observed differences in
ER and PR reactivity in samples stored in similar conditions
(4�C) for several days.5 Enzyme-rich tissues, such as intes-
tine or pancreas, autolyze rapidly; proteolysis can lead to
increased background staining (Fig. 2).149 Diffusion of
Table 1. Steps and Variables in an Immunohistochemical Test.
Steps Variables
Preanalytical phase Sample procurement Delayed fixation, prolonged ischemia, thickness of sampleFixation Cross-linking vs coagulating fixatives, durationDecalcification Type of decalcification solution and durationTissue processing Paraffin-embedded vs frozen tissuesTissue sectioning Thickness of tissue section, drying temperature and duration, tissue section aging
Analytical phase Deparaffination Dewaxing agentAntigen retrieval Detergents, enzymes, HIERBlocking nonspecific
reactivitiesEndogenous enzymes, hydrophobic binding, pigments
Primary antibody Monoclonal vs polyclonal, Ag recognition (native vs linear), specificity, speciesvariability
Detection system Avidin-biotin vs polymer-based systems, ultrasensitive methodsEnzyme-substrate-
chromogenColor detection
Multiplex IHC Enzyme-substrate combinationsCounterstain Contrast between chromogen and counterstain
Postanalytical phase Control performance Animal species compatibility, tissue processingInterpretation Pathologist vs automated evaluationReport Percentage of positive cells, positive vs negative threshold, stand-alone test vs
ancillary testDiagnostic, prognostic, or theranostic test
HIER, heat-induced antigen retrieval; IHC, immunohistocehmistry.
44 Veterinary Pathology 51(1)
soluble proteins with fixation delay has been reported with
thyroglobulin, myoglobin, glial fibrillary acidic protein
(GFAP), and other cellular proteins.95,184,355 Protozoa
and fungi may be more resistant to autolysis than viruses.291
If delay in fixation is anticipated, samples should be
refrigerated.149
The quality of fixation is influenced by the type of fixa-
tive; fixative pH, buffers, concentration, osmolality, and
additives; fixation time and temperature; and the use of
postfixation procedures (eg, decalcification). Two types of
fixatives are used in histopathology: cross-linking (noncoa-
gulating) and coagulating. The choice of fixative determines
the need for pretreatments (eg, antigen retrieval), titer of the
primary antibody, the intensity of the specific reaction ver-
sus background (signal-to-noise ratio), and even the detec-
tion pattern of antigens.149,264
Formaldehyde: A Cross-Linking Fixative. Formaldehyde is the
standard fixative for routine histology and IHC. Two factors
contribute to the use of formaldehyde as the fixative of choice
for most histologic procedures: (1) for more than a century,
pathologists have studied tissues fixed in formalin and have
become accustomed to histologic examination through the
artifacts it produces, and (2) archived paraffin blocks, an
invaluable resource for disease studies, most commonly con-
tain formalin-fixed tissues.85 Formaldehyde preserves mainly
peptides and the general structure of cellular organelles. It also
interacts with nucleic acids but has little effect on carbohy-
drates.91,194 It is a good preservative of lipids if the fixative
contains calcium.176
The Chemistry of Formalin Fixation. Fixation with formaldehyde
is a 3-step process of penetration, covalent bonding, and
cross-linking.51,77 Formation of cross-links between target
peptides and irrelevant proteins reduces or blocks their
immunoreactivity.39 Whereas these steps happen simultane-
ously, they do so at different rates, with penetration being
about 12 times faster than bonding and the latter 4 times faster
Table 2. Comparison of Fixatives Used in Immunohistochemistry.
Fixative Description Fixation Times Advantages Disadvantages
Formalin 10% neutral bufferedformalin
>8 hours Most common fixative Fixation (cross-linking) is slow
12–36 hours necessary forcomplete fixation
Quick penetration Ag cross-linking results in need forAg retrieval
Special handling and disposalrequirements
Zincformalin
Mixture of zinc sulfateand formalin
4–48 hours Shorter fixation times thanformalin
Possible quenching of primaryfluorescence
May not need antigenretrieval
Special handling and disposalrequirements
Preserves tissue morphologywell
Alcohol/acetone
70%, 90% EtOH or90% EtOH/10%acetone
Variable; often occurs intissue processingsecondary to formalinfixation
Shorter fixation times Unknown archival stability
10–15 minutes for cryostatsections/cytology smears
Good preservation ofcytoplasmic intermediatefilaments
Shrinking or hardening of the tissue
Bouin’s Mixture of formalinand picric acid
1–12 hours Rapid tissue fixation Reduced preservation of manyantigens, particularly lipid-containing antigens
Useful for endocrine tissuesand tumors
Fixation may result in brittle tissuesdifficult to section
Postfixation procedure to removepicric acid
Needs special handling and disposalrequirements
B-5a Mixture of mercuricchloride, sodiumacetate, and formalin
1–6 hours Used mostly in lymphoidtissue due to enhancedcytological detail andimmunoreactivity ofimmunoglobulins andintracytoplasmic antigens
Tissue hardeningSurface immunoglobulins not well
preservedSpecial handling and disposal
requirements
Modified and used with permission by the Clinical and Laboratory Standards Institute, document I/LA28-A2.149
aFixatives containing mercury require special handling and disposal procedures.
Ramos-Vara and Miller 45
than cross-linking.51 For example, a 3-mm-thick sample will
be 100% penetrated, 24% bonded, and 6% cross-linked in 8
hours; after 24 hours of fixation, it will be 70% bonded and
36% cross-linked.53 Fixation at 37�C is faster than at
25�C.145 Notably, formaldehyde penetrates tissues rather
quickly, but tissue penetration does not equal tissue fixation.
Tissue fixation by formaldehyde is considered a clock
reaction because formaldehyde is in equilibrium with methylene
glycol.149 Only formaldehyde (and not methylene glycol) pro-
duces the characteristic cross-linking formaldehyde fixa-
tion.109,149 As formaldehyde is used during tissue fixation,
methylene glycol is converted into formaldehyde to maintain
the equilibrium; this newly formed formaldehyde reacts with
proteins, producing additional cross-links and therefore further
fixation.149 In solution, formaldehyde binds the following
amino acids: lysine, tyrosine, asparagine, tryptophan, histidine,
arginine, cysteine, and glutamine.235,261,334 Therefore, knowing
the amino acid composition of an epitope may predict its sen-
sitivity to fixation in formalin.348 However, the cross-linking
between antigens and unrelated tissue proteins also has a
marked effect on the immunoreactivity of antigens after
fixation.347 Cross-linking due to aldehyde fixation can produce
new epitopes that specifically react with antibodies designed
for another purpose (eg, the specific detection of enamel
proteins in glutaraldehyde-fixed tissue by an antibody to
vimentin).178
The basic mechanism of fixation with formaldehyde is the
formation of addition products (adducts) between the formalin
and uncharged reactive amino groups (-NH or NH2) that even-
tually will form cross-links.75 The formation of methylol
adducts inactivates nucleases and proteases.261 Once the
addition product (reactive hydroxymethyl compound) is
formed, additional cross-links develop. Thus, in the presence
of a second reactive hydrogen, the hydroxymethyl group forms
a methylene bridge.
1. Formation of addition products
Protein-H + Protein-CH2OH
Reactive hydrogenon tissue
Formaldehyde Reactive hydroxymethylcompound
addition product
CH2O
2. Formation of methylene bridges
2Protein-CH OH + Protein-H
Reactive hydroxymethylcompound
addition product
Second reactive hydrogenon the protein
Protein-CH2-Protein + 2O
Methylene bridge cross-link
H
The final result of formaldehyde fixation is a profound change
in the conformation of macromolecules, which may make the
recognition of proteins (antigens) by antibodies difficult or
impossible.245 Although the primary and secondary structures
of proteins are relatively spared, formalin fixation alters the 3-
dimensional (tertiary and quaternary) structure of pro-
teins.142,231 Changes in the tertiary structure may not be the
direct result of formaldehyde fixation but rather the subsequent
interaction of cross-linked proteins with ethanol or clearing
agents during tissue processing.77,108,261 More energy (eg,
antigen retrieval) is needed to reverse formalin fixation
cross-links after processing tissues in ethanol, confirming that
antigen immunoreactivity is affected not only by formalin fixa-
tion but also by postfixation procedures.108
The deleterious effects of formaldehyde on immunoreac-
tivity in cells and tissues can be partially reversed,92 whereas
glutaraldehyde fixation is considered irreversible.91 Pro-
longed washing of fixed tissues reduces further fixation by
removing unbound formaldehyde,142 although established
cross-links remain.92 Long-term storage of formalin-fixed
tissues in alcohol halts the formation of cross-links and, there-
fore, facilitates antigen detection should the tissues be needed
subsequently for IHC. Overfixation can also be partially cor-
rected by soaking tissues in concentrated ammonia plus 20%chloral hydrate.210
The pH of a fixative buffer dramatically influences the
degree of cross-linking.92,113,282 Amino acids are charged
(-NH3þ) at lower pH and uncharged (-NH2) at higher pH.
When using neutral buffered formalin, the pH is shifted to
neutrality, causing dissociation of hydrogen ions from the
charged amino groups (-NH3þ) of the protein side chains,
resulting in uncharged amino groups (-NH2). These
uncharged groups contain hydrogen ions that can react with
formalin to form addition groups and cross-links. In other
words, the use of 10% buffered formalin (pH 6.8–7.2) pro-
duces more cross-links than nonbuffered formalin and there-
fore more deleterious effects for IHC.284 If the fixative is
acidic formalin, cross-linking is reduced, so the antigen retrie-
val procedure may need modification. In comparison, 10%neutral buffered formalin, 10% formal saline, and 10% zinc
formalin were each superior to 10% formal acetic in preser-
ving antigenicity.412
After an initial drop in immunoreactivity with fixation, there
is a plateau of variable duration before antigens become unrec-
ognizable by specific antibodies.347 Overfixation could produce
false-negative results due to excessive cross-links,142 especially
before the advent of heat-based antigen retrieval methods.205
However, significant reduction of immunoreactivity is detected
in only a few markers after several weeks’ fixation (Fig.
3),238,403,404 so it seems that the negative effects of overfixation
are antigen and antibody dependent and can be overcome in
many instances with an appropriate antigen retrieval proce-
dure.6,22,164,242 Vimentin was once used as an internal control
of antigenicity loss in overfixed tissues.18,306 However, with
heat-based antigen retrieval, some antigens in overfixed tissues
may not be well preserved even if vimentin reactivity does not
indicate antigen degradation.6 The effect of prolonged
formaldehyde fixation on an antigen depends on its cellular loca-
lization142—for example, there is irreversible loss of Bcl-2
46 Veterinary Pathology 51(1)
immunoreactivity in the nucleus, even with heat-based antigen
retrieval, whereas cytoplasmic immunoreactivity is preserved
or increased after antigen retrieval.154
Underfixation can also produce unexpected results and is
considered a more common and serious problem than overfixa-
tion.35,149 In a typical diagnostic setting, formalin-fixed tissues
are processed through a series of alcohol gradients prior to
paraffin embedding. If large samples are fixed only 24 to 48
hours or small biopsy specimens for only several hours,
cross-links may develop only in the periphery of the specimen
with the core of the tissue left unfixed or fixed only by coagu-
lation with the alcohol used for dehydration during tissue
processing,142,405 resulting in a IHC gradient across the section
(Fig. 4).149 Underfixation can produce irrelevant or equivocal
results that can alter the detection or scoring of some biomar-
kers.123,171 Antigen retrieval of underfixed tissues may result
in unexpected antigen detection.284 Underfixed tissues are
easily damaged by harsh antigen retrieval methods, with subse-
quent loss of antigenicity.303
There is no optimal fixation time for every antigen. The
structure of the antigen as well as its relationship with other
proteins probably influences the effect of fixation on immu-
noreactivity. The current Clinical and Laboratory Standards
Institute recommendation for formalin fixation of diagnostic
specimens is 16 to 32 hours; on average, complete fixation
is achieved after 24 to 48 hours.149 Interestingly, the recom-
mended 10:1 ratio of fixative to sample was challenged when
a 2:1 ratio and 48-hour fixation at room temperature was
considered sufficient for routine histology and IHC.53 Fixa-
tive penetration, bonding, and cross-linking are faster at
higher temperatures. Samples to be fixed should not be
thicker than 2 to 4 mm. Although formaldehyde has been
deemed a suboptimal fixative for IHC, with optimal antigen
retrieval procedures, it is satisfactory for most antigens.
Glyoxal, the Ideal Formalin Substitute? Glyoxal is a dialdehyde
that resembles 2 back-to-back formaldehyde molecules.76
Whereas formaldehyde forms a single hydrated species
(methylene glycol), glyoxal has several hydrated forms, the
most common of which is 1,3-dioxolane.76 One advantage of
glyoxal over formaldehyde is that it does not emit vapors. In
addition, glyoxal has poor reactivity toward most end groups
and forms addition compounds only with arginine, lysine,
cysteine, and the single a-amine group of proteins, whereas
formaldehyde reacts at room temperature (RT) with almost all
end groups that contain nitrogen or oxygen to form single-
carbon adducts.76 In comparison to formaldehyde fixation,
glyoxal fixation is faster with minimal or no cross-linking,
rendering antigen retrieval unnecessary for many antigens.
Although a special antigen retrieval solution with high pH can
be used, standard antigen retrieval procedures in glyoxal-
fixed tissues may be ineffective or even deleterious.76 The
perceived advantages of glyoxal were disputed, however, in
a quantitative image analysis study in which formaldehyde
was considered superior in terms of morphologic preservation
and IHC signal.59
Coagulative Fixatives. The problems with formaldehyde fixation,
especially the loss of immunoreactivity (particularly before the
development of heat-based antigen retrieval methods), have
prompted a search for alternative fixatives.284 Many formalin
substitutes are coagulating fixatives that precipitate proteins
by breaking hydrogen bonds without protein cross-linking. The
most common types of coagulative fixatives are dehydrants
(alcohols and acetone) and strong acids (picric acid, trichloroa-
cetic acid). Most body fluid proteins have hydrophilic moieties
in contact with water and hydrophobic moieties in closer
contact with each other, stabilizing hydrophobic bonding.
Removal of water by ethanol destabilizes protein hydrophobic
bonding because the hydrophobic areas are released from the
repulsion of water, and the protein tertiary structure unfolds
(Fig. 5).92 Simultaneously, removal of water destabilizes
hydrogen bonding in hydrophilic areas. The resulting protein
denaturation causes inadequate cellular preservation142,153,401
and a possible shift in intracellular immunoreactivity as
reported for some growth factor peptides and cytokines
(Fig. 6).42,383 Coagulative fixation maintains tissue structure
at the light microscopic level fairly well but results in cytoplas-
mic flocculation as well as poor preservation of mitochondria
and secretory granules.284
The Search for Alternatives to Formaldehyde Fixation. There is no
‘‘one fixative fits all’’ for IHC.128 Formaldehyde is used mainly
because it is reliable for general histology, it is inexpensive,
and its deleterious effects can often be countered with antigen
retrieval.280 In addition, the interpretation of retrospective
studies would be onerous if archived paraffin blocks contained
tissues preserved in a variety of fixatives over the years. The
value of nonformaldehyde fixatives for research purposes has
been demonstrated for various antigens.* Most available
formalin substitutes are alcohol solutions; therefore, fixation
is based on dehydration and protein coagulation.51,83,198
Weigners fixative, a mixture of alcohols and pickling salt,
appears to be superior to formaldehyde for DNA and RNA
studies, performed comparably to formaldehyde for IHC, and
maintained immunoreactivity for some biomarkers after
prolonged fixation when formaldehyde did not.193 Carnoy’s
fixative, a mixture of alcohol–chloroform–acetic acid, was
superior to formalin for IHC detection of some antigens
without antigen retrieval in tissue-engineered constructs.195
Tissues fixed in PAXgene, which consists of a fixation reagent
(methanol, acetic acid, and a organic solvent) and a postfixa-
tion stabilization reagent (alcohol mixture), may not need anti-
gen retrieval for some antigens,183 although immunoreactivity
for other antigens is reduced compared with formalin-fixed,
paraffin-embedded (FFPE) tissues.21 For molecular analysis,
PAXgene preserves RNA better than does formalin.130
The immunoreactivity of antigens in fixed and paraffin-
embedded tissue varies markedly depending on the antigen and
*References 75, 91, 103, 118, 171, 193, 198, 241, 244, 262, 280, 317, 322, 352,
432, 434.
Ramos-Vara and Miller 47
more specifically on the epitope recognized by the antibody.8,59
In a comparison of the effect of different fixatives (strong
cross-linkers, weak cross-linkers, coagulant, and combination
coagulant/cross-linkers) on antigen immunoreactivity in mouse
tissue, formaldehyde, followed by the combination fixatives,
performed better than the other types.59 Substitution of a differ-
ent fixative in an IHC test validated for formalin-fixed tissue
demands a new validation study.149
Microwave Fixation. The use of a microwave can reduce fixation
time and therefore the overall time for tissue processing. After
immersing the tissue in formalin at RT for at least 4 hours,
adequate fixation can be achieved in a 5-mm-thick sample by
microwaving in formalin solution for 1.5 to 4 minutes at
55�C.149 Validation of microwave fixation procedures for IHC
mandates the use of tissue controls processed in a similar way.
Microwave procedures require approved laboratory micro-
waves and, because of the release of formalin vapors, must
be performed in a fume hood.
Decalcification
Decalcification with weak acids does not seem to interfere sig-
nificantly with IHC for most antigens, provided the tissues
were well fixed in formalin;284 however, decalcification with
strong acid solutions has a negative effect on immunoreactiv-
ity, at least for some antigens.7,9,96,232 In a study of melanocytic
markers in canine tissues, decalcification for less than a week
with formic acid did not significantly reduce immunoreactivity
for Melan A, PNL2, or tyrosinase, whereas the use of HCl in
the decalcifying solution reduced the immunoreactivity for
Melan A and PNL2 after only 1 day of treatment with complete
loss of immunoreactivity after 1 week.295 In that study,
tyrosinase was more resistant than the other 2 markers to the
deleterious effects of HCl decalcification. The antigenicity of
intermediate filaments appears to be more resistant to decalci-
fication effects than other antigens.149 Antigen retrieval in a
boric acid solution at 60�C seems to improve the immunoreac-
tivity of some antigens in decalcified tissues.414 In summary,
weak (eg, formic) acid decalcifying solutions diluted in
formalin are recommended for IHC. Due to the potential neg-
ative effects of decalcifying solutions, the IHC report should
indicate the type, if any, of decalcification.
Tissue Processing and Incubation Buffers
Although fixation is paramount in the outcome of the antigen-
antibody reaction, the incubation buffer and tissue-processing
solutions can also alter antigenicity.89,143 The combination of
cross-linking fixatives with heat and the nonpolar solvents used
in paraffin embedding is thought to modify the antigen confor-
mation so that specific epitopes may not be recognized by
antibodies that would recognize those epitopes in frozen
sections. Thus, fixation may not be the sole factor in failure
to detect an antigen.89,113,129 There is also evidence for a cumu-
lative effect of fixation and tissue-processing factors in the
failure of antigen recognition in FFPE from a study of Ki67 and
proliferating cell nuclear antigen (PCNA) immunoreactivity in
formaldehyde-fixed tissues processed with alcohol and
xylene.77,264 Shifts in the tertiary structure of proteins so that
hydrophobic areas are oriented outward and hydrophilic
regions inward (the so-called hydrophobic inversion)77 during
dehydration and clearing steps of tissue processing can reduce
or abolish antibody binding without antigen retrieval, espe-
cially with poorly stabilized (unfixed or suboptimally fixed
in formalin) tissues/proteins exposed to a weakly polar or
nonpolar solvent.264 This negative effect varies with the
dehydrating and clearing agent used.93 There is also increased
background reactivity in tissues left in xylene for prolonged
periods during processing.149
Few authors have evaluated the effects of long-term storage
of formalin-fixed tissues in other solutions.93,359,363 Formalin-
fixed tissues stored in 70% ethanol for several weeks had no
apparent loss of immunoreactivity or nucleic acid amplifica-
tion.359 Tissues stored in tap water for up to a week had no loss
of antigenicity or changes in microscopic appearance.363
The effects of dewaxing with organic solvents on IHC have
not been studied systematically. However, nonorganic paraffin
solvents (eg, dishwashing detergents) have been substituted for
organic solvents with comparable results.52,148
Tissue Microarrays
Tissue microarrays (TMAs), introduced by Kononen et al,197
allow simultaneous examination of hundreds of tissues on a
single microscope slide. A tissue core is transferred from the
‘‘donor’’ paraffin block to the ‘‘recipient’’ block, which may
contain up to 1000 cores.344 This technology is used not only
for protein detection but also for gene expression.y Tissue
microarray techniques include multitumor arrays (samples
from tumors of multiple histological types), progression arrays
(samples of normal tissue and different stages of tumor
progression within a given organ), prevalence arrays (tumor
samples from 1 or several entities without extensive clinico-
pathologic information to test biomarkers with possible thera-
peutic implications), prognosis/outcome-based arrays (samples
with clinical follow-up data to evaluate prognostic or predictive
biomarkers), cell line arrays (normal or cancer cell lines grown
in culture to determine the specificity of antibodies targeting a
protein), heterogeneity/random tissue/tumor arrays (tumor and
nontumor tissues from different sites for monitoring the
intratumoral heterogeneity of molecular markers), and cryomi-
croarrays (frozen samples that might be more suitable than
formalin-fixed tissues for RNA detection).256,316,324,344,367
The advantages of the TMA method include less reagent con-
sumption; decreased technical time; decreased variability of
results; the possibility of digitizing and quantifying results or
interpretation of results by hierarchical cluster analysis, quality
control, and standardization; evaluation of antibody sensitivity
yReferences 120, 121, 146, 172, 243, 256, 258, 266, 316, 346, 381.
48 Veterinary Pathology 51(1)
and specificity; and rapid and high-throughput discovery and
validation of biomarkers.173,258,305,311,358 With an adequate selec-
tion of control tissues, fewer than 12 tissue cores in an array
suffice to evaluate more than 90% of the markers used in diagnos-
tic IHC.150 With tissue microarray, the cell expressing a particular
gene can be identified, whereas in DNA microarray, the sample is
digested before testing, so cell expression cannot be localized.
Although the concept of tissue microarray is simple, this method
has some disadvantages compared with the classic single sample/
microscope slide: preparation (with a commercial manual or
automated array) requires high technical skills and careful plan-
ning, sample selection is critical due to its small size and the usual
heterogeneity of the sample, and differences in fixation protocols
in archival tissue can influence TMA results.99,172,173,266,358
Sampling of multiple and different areas of the donor block
using small (0.6-mm) cores can be more representative of the
lesion and less likely to damage the block than extracting a sin-
gle and larger core.99,324 Results from triplicate TMA cores had
up to 98% concordance with the results from full-block sections,
whereas concordance was lower with only 1 or 2 cores.99,119
Nevertheless, intratumor heterogeneity can be a complicating
factor when assessing biomarkers in TMAs,212 especially when
a scoring system (eg, 0, 1þ, 2þ, 3þ for HER2 in breast cancer)
for the IHC reaction is used for therapeutic planning.
The current Clinical and Laboratory Standards Institute
(CLSI) guidelines for IHC recommend TMAs for internal and
external quality assurance programs. However, due to various
technical issues, the use of TMA for diagnostic purposes is not
recommended.125,149 The amount (diameter and number of cores)
of tissue needed to test a given biomarker varies and should be
based on the type of tissue/disease process and biomarker
examined.149
Multiplex immunostaining (MI) chips can be used to examine
multiple antigens in the same tissue section. MI chip technology
differs from tissue microarray in that MI permits the analysis of
expression of as many as 50 antigens in a single specimen,
whereas the microarray technology permits the analysis of a sin-
gle antigen in many specimens simultaneously.117 The main
problem in applying this technology in a clinical setting is the het-
erogeneity of most tumors and, therefore, the possibility of false-
negative results.117
Drying of Paraffin Sections
Paraffin sections dried overnight at temperatures of 60�C or
higher may have reduced immunoreactivity for some anti-
gens.93,137,147,412 In another study, immunoreactivity was
reduced when slides were dried at temperatures higher than
68�C for more than 16 hours, whereas drying at 58�C for 24
hours did not affect immunoreactivity.177
Storage of Paraffin Blocks and Tissue Sections
Paraffin blocks containing formalin-fixed tissues are a power-
ful resource for protein and nucleic acid investigations.192 They
are the archived tissue most suitable to evaluate diseases and
apply the results to targeted medicine.354 It is the authors’ and
others’327 opinion that paraffin blocks remain stable for years
in terms of antigenicity. However, only controlled studies on
the effect of nucleic acid detection in archival paraffin blocks
have been published.133 Any deleterious effects of prolonged
paraffin block storage on tissue antigenicity could differ among
antigens and should be considered if IHC results on old paraffin
blocks are inconsistent or unexpected.
Storage of unstained paraffin control tissue sections increases
efficiency but may adversely affect immunoreactivity (tissue sec-
tion aging/slide oxidation/biomolecule degradation).93,127,149
These differences are epitope specific rather than protein target
specific.149 Both light and temperature contribute to the loss of
antigenicity that occurs with photo-oxidation of tissue sec-
tions.30,304 Tissue section aging is a rather common problem with
nuclear antigens (eg, Ki67, ER, p53);z however, in a study of ani-
mal tissues, cytoplasmic membrane antigens were more sensitive
than nuclear antigens to degradation from photo-oxidation and/or
ambient temperature.304 Immunoreactivity can also decrease
when tissues are suboptimally dehydrated (retained endogenous
water) during paraffin embedding or with paraffin sections stored
under high humidity.420 To reduce antigen degradation due to
photo-oxidation, paraffin sections should be used within 1 week
of sectioning or at least should be stored in an airtight container
in the dark.149 Keeping the paraffin sections in the dark under
refrigeration also diminishes the loss of antigenicity.304
In summary, all steps in tissue processing, from acquisition
to storage, can affect the quality of the IHC test.376 When there
is decreased intensity or loss of reaction in stored control tissue
sections, the IHC test should be repeated with a different stored
control tissue section plus a freshly cut control tissue section. If
the change is limited to stored sections, any unused stored
control slides should be discarded.
Analytical Phase of IHC
Antigen Retrieval
Fixation and tissue processing modify the 3-dimensional
structure of proteins, which can render antigens undetectable
by specific antibodies because the immunologic reaction
depends on the conformation of the antigen.143 The purpose
of antigen retrieval (AR) procedures is to reverse the changes
produced by fixation. AR is particularly important for tissues
fixed in cross-linking fixatives. Approximately 85% of anti-
gens fixed in formalin require some type of AR to optimize the
immunoreaction.287 The need for AR and choice of method
depend on the targeted antigen and the type of antibody;393
AR is more commonly necessary with MAbs than with
PAbs.287 The 2 most common AR procedures in IHC are
enzymatic and heat-based retrieval. Although AR facilitates
detection of Ags, background staining or antigen detection in
zReferences 26, 81, 133, 169, 219, 233, 260, 281, 303, 397, 406.
Ramos-Vara and Miller 49
unusual locations is not uncommon with harsh AR methods and
can preclude diagnostic interpretation (Fig. 7).
Antigen Retrieval With Enzymes. Protease-induced epitope
retrieval (PIER), introduced in the mid-1970s,161,162 was the
most common AR method before the advent of heat-based
AR in the 1990s. Commonly used enzymes include trypsin,
proteinase K, pronase, ficin, and pepsin.20,170,236,263,273 The
mechanism of PIER is probably protein digestion, but this clea-
vage is nonspecific and some antigens may be negatively
affected.390 The effect of PIER depends on the concentration
and type of enzyme, incubation parameters (time, temperature,
and pH), and the duration of fixation.20,236,273 The enzyme
digestion time is proportional to the fixation time.390 It is
practical to optimize a few enzymes for laboratory use. A com-
mercially available ready-to-use solution of proteinase K has
good activity at room temperature, so can be used with
automatic stainers. The disadvantages of PIER are the low num-
ber of antigens for which it is the optimal AR method and the
risk of altering tissue morphology or destroying epitopes.263,390
Interestingly, proteinase treatment has facilitated IHC detection
of nucleolar proteins present in high concentration but
undetectable without AR, whereas nucleolar proteins at low
concentration can be detected without enzymatic treatment.364
Heat-Induced Epitope Retrieval. Heat-induced epitope retrieval
(HIER) has revolutionized the IHC detection of antigens fixed
in cross-linking fixatives. In addition, HIER is used for
extraction of molecules (eg, nucleic acids, proteins) from
formalin-fixed, paraffin-embedded tissues.332 HIER was intro-
duced by Shi et al.337 based on a concept developed by
Fraenkel-Conrat et al110–112 that the chemical reactions
between proteins and formalin could be reversed (Fig. 8), at
least in part, by high-temperature heating or strong alkaline
hydrolysis. Although HIER denatures formalin-fixed tissues,
it paradoxically restores their immunoreactivity.348 The
mechanism involves the dissociation of irrelevant proteins
from target peptides.39 The secondary and tertiary structure
of proteins is probably modified (denatured) during HIER;
however, that should not affect the immunoreactivity of most
antigens because IHC antibodies require only an intact primary
(linear) protein structure.39,347 Likewise, some comformational
epitopes become denatured after HIER and will not bind
specific antibodies.421 Heating may unmask epitopes by hydro-
lysis of methylene cross-links.135,390,422,423 Rupture of cross-
links resets the charged status of proteins and their hydrophobic
characteristics.421 AR also acts by other mechanisms because it
enhances immunoreactivity of tissues fixed in ethanol, which
does not produce cross-links.144 Even in unfixed tissues, anti-
gens can be unmasked by HIER, perhaps because steric barriers
produced by the antigen itself had precluded antibody access to
targeted intramolecular epitopes.180
Other hypotheses to explain HIER are extraction of diffusible
blocking proteins; precipitation of proteins; rehydration of the
tissue section, allowing better antibody penetration; and heat
mobilization of trace paraffin.349 Tissue-bound calcium ions
may mask some antigens during fixation. Calcium chelating sub-
stances (eg, EDTA) are sometimes more effective than citrate
buffer in AR.247,248,271,415 Calcium-induced changes in protein
conformation may augment the immunodetection of some anti-
gens,399 but for many antigens, calcium-induced effects on
immunoreactivity cannot be documented.331,333 Restoring the
native electrostatic charges modified during formalin fixation
has also been considered an AR mechanism.34,36
Equipment used for HIER includes decloakers (commercial
pressure cookers with electronic controls for temperature and
time), vegetable steamers, water baths, microwave ovens, or
pressure cookers.13,88,182,271,337 The relationship between
temperature and exposure time is inverse: the higher the
temperature, the shorter the time needed to achieve results.
Heating at high temperature (100�C) for a short duration (10
minutes) is better than heating at a lower temperature for a lon-
ger time.143 In most instances, the source of heat is not critical
to the outcome but a matter of convenience. Satisfactory results
can be obtained using a steamer (90�C–95�C) for 20 minutes
for most antigens, although a decloaker eliminates the
problems of irregular heating or temperature variation with a
steamer or microwave oven.
A universal antigen retrieval solution is not available.143,167
Thus, several HIER solutions made of different buffers (eg,
citrate, tris, Tris-HCl) and pH (3–10) have been used. Some
antigens can be retrieved with low pH solutions, others only
with high pH solutions, and a third group with solutions of a
wide pH range.332,336 The importance of the chemistry of the
retrieval solution, particularly the buffer, is unclear; however,
for most antigens, HIER with 0.01 M sodium citrate buffer
(pH 6.0) produces satisfactory results with good cell morphol-
ogy when compared with buffers with higher pH or solutions
containing EDTA.19,87,143 However, use of different buffers
may be required to optimize antigen recovery. A low pH buffer
(acetate, pH 1.0–2.0) appears especially useful for nuclear
antigens. As a rule, high pH and EDTA-containing buffers
reach their optimal treatment effect faster than do lower pH
citrate-containing buffers.199 Sometimes multiple AR methods
(enzyme-HIER; HIER-HIER) are needed to optimize the
immunodetection of antigens (Fig. 9).97,143,179,340,388 Triple
AR was beneficial to demonstrate amyloid b.179
The degree of fixation can dramatically modify the response
of antigens to AR. Unfixed proteins are denatured at tempera-
tures of 70�C to 90�C, whereas formaldehyde-fixed proteins
are resistant at the same temperatures.231 This could explain
why the immunoreactivity of partially fixed tissue can be
heterogeneous, despite the supposed even distribution of the
antigen.19 In other words, formaldehyde fixation may protect
against denaturation during AR, although this has been dis-
puted, at least when using an autoclave reaching 120�C.39,348
For practical purposes, protein denaturation during AR does
not have a negative impact on the immunoreactivity of peptides
(Fig. 10).
The variability in both the kinetics of fixation and AR for
different epitopes suggests that different degrees of cross-
linking may occur depending on the peptide amino acid
50 Veterinary Pathology 51(1)
Figure 6. Effects of fixation on small peptides and cell membrane antigen stability. Formalin fixation stabilizes small peptides and cell membrane anti-gens, preventing leakage or diffusion, even after sectioning. With acetone fixation, small peptides leak through the permeabilized cell membrane. For-malin fixation stabilizes membrane antigens in cryostat sections, but small peptides leak away. Used with permission and modified from Floyd and vander Loos.106 Figure 7. Effects of antigen retrieval (AR) on immunohistochemistry (IHC). Esophagus; dog. (a) No AR. (b) Proteinase K (PK)AR. (c) Heat-induced epitope retrieval (HIER) with citrate, pH 6.0. With no AR, myoglobin expression is observed as expected in skeletal muscle
Ramos-Vara and Miller 51
composition.39 The possibility of unexpected immunoreactiv-
ity should always be considered when using HIER, particularly
with low pH buffers.19 When practical, the immunoreactivity
of fresh-frozen and routinely processed paraffin tissue sections
should be compared331 because not all antigens benefit from
AR, even after prolonged formalin fixation.349
Miscellaneous Antigen Retrieval Methods. Pretreatment with
concentrated formic acid improves the signal in some IHC
tests.189 Other AR methods include the use of strong alkaline
solution, urea, borohydride, and a solution of sucrose or acid
(eg, hydrochloric acid).330,334 Citraconic acid with heat has
been used as AR to reverse formalin-induced cross-linking,
presumably by hydrolysis.249,253 This method has been
successfully applied to nervous tissue fixed more than 5 years.4
The proposed mechanism of citraconic anhydride (citraconic
acid) AR is converting the cross-links in the sample to pro-
tected amines and liberating formaldehyde quickly to avoid
reforming adducts; then citraconic acid liberates amines via
hydrolysis.253
Detergents, although not strictly speaking an AR compo-
nent, facilitate Ag-Ab binding by solubilizing membrane
proteins.303 Detergents form mixed micelles with lipids as well
as micelles that contain proteins (also known as surfactants),
thereby decreasing the surface tension of water. They are usu-
ally incorporated into the dilution/rinse buffers. Examples of
detergents in IHC include ionic detergents, bile acid salts, and
nonionic and zwitterionic types (eg, Triton R-X100, BRIJR,
Tween 20, saponin). The addition of sodium dodecyl sulfate
(SDS) to the AR buffer and HIER at relatively low temperature
(97�C) appears to improve the signal of many markers.366
All-in-One Epitope Retrieval Buffers. The classic histologic proce-
dure calls for dewaxing paraffin sections using organic solvents
(eg, xylene) followed by dehydration in alcohols and hydration
before AR. The so-called 3-in-1 reagents can perform aqueous
deparaffination, rehydration, and AR.149 Their use in IHC is
relatively new, so comparison with traditional means of depar-
affination, rehydration, and AR is required before using them in
a diagnostic setting. Commercially available all-in-one epitope
retrieval buffers can reduce processing time by melting the
paraffin in tissue sections during HIER.148 The advantage of
these solutions is obvious, but reported instances of incomplete
paraffin removal could affect the outcome of the IHC.114 This
is more commonly observed with solutions based on surfac-
tants and wetting agents, which tend to incompletely solubilize
the molten paraffin. The addition of a water-soluble organic
agent appears to solve this problem without negatively affect-
ing the IHC reaction.114
Antigens and Antibodies: The Specificity Issue
Immunohistochemistry involves antibody recognition and
binding to an epitope on the target antigen.149 The specificity
of the reaction largely depends on qualities of the primary
antibody and the ability of the antigen (epitope) to bind it. The
most commonly used immunoglobulin (Ig) is IgG; IgM is used
less commonly.284
Antigens. Antigens can be as small as a monosaccharide;
epitopes consist of 5 or 6 amino acid residues.149 Antigens can
be multivalent with multiple identical epitopes (homopoly-
meric) or multiple distinct epitopes (heteropolymeric).213 A
single gene can generate several different antigen isoforms via
2 principal mechanisms. Alternative splicing of the primary
gene transcript can produce multiple different mature
transcripts, each of which codes for a slightly different
protein.44,328 Many proteins also undergo posttranslational
modifications, such as glycosylation, phosphorylation, and
proteolytic processing, which add further complexity.237 As a
result, 1 gene can generate numerous protein (antigen) iso-
forms, and this repertoire can change with time (eg, tenascin
and hemoglobin isoforms change from fetal development to
adulthood).237
Two broad groups of immunogens are used to produce
antibodies: synthetic peptides and purified protein prepara-
tions.237 The known amino acid sequence of synthetic peptides
facilitates IHC interpretation, both with respect to the isoforms
of the target protein and any cross-reactivity with similar pep-
tide sequences in other proteins. A potential disadvantage to
using synthetic peptides is that an isolated synthetic peptide
sequence may lack the normal 3-dimensional structure of the
native protein. In addition, other proteins can be intimately
associated with the protein of interest in vivo. Either factor can
mask target epitopes, preventing their detection by antibodies
raised to synthetic peptides and therefore yielding false-
negative results. Also, crucial in vivo posttranslational modifi-
cations of the native antigen are absent in synthetic pep-
tides.220,221 The presence of the synthetic peptide sequence in
unrelated proteins could produce immunologically specific
Ag-Ab binding (molecular mimicry) despite the absence of the
target antigen.29,72,165 Use of purified proteins as immunogens
avoids many of the problems of synthetic peptides.237 How-
ever, protein purification to homogeneity from cells or tissues
can be technically difficult, and contaminating proteins may be
Figure 7. (continued) (solid circle) with no labeling of mucosal epithelium (arrowhead), plasma (V), or smooth muscle (asterisk). With PK AR,background reactivity develops in mucosal epithelium, plasma, and smooth muscle. Nonspecific labeling with HIER, in contrast to that with PK,has a predominantly nuclear pattern in epithelium and smooth muscle (inset). Immunoperoxidase-DAB for myoglobin, hematoxylin counterstain.Figure 8. Conformational changes in protein structure from cross-linking fixatives. Modification of epitope (colored segments) presentation couldpreclude access by specific antibodies. Fixation-induced conformational changes can be reversed by HIER. Figure 9. Antigen retrieval. Kidney; dog.Collagen type IV was demonstrated by sequential double AR with HIER (citrate) and pepsin. No reactivity with pepsin AR alone (lower inset) or HIERalone (upper inset). Immunoperoxidase-DAB, hematoxylin counterstain.
52 Veterinary Pathology 51(1)
Figure 10. Loss of immunoreactivity after formalin fixation with recovery by antigen retrieval (AR). (a) Putative structural changes in nativeproteins. (b) Corresponding changes to epitopes in the in vitro peptide assay. Used with permission from Sompuran et al.347
Ramos-Vara and Miller 53
more antigenic than the protein of interest, producing a dispro-
portionate and unwanted immunogenic response. Another
problem with purified proteins arises if the targeted antigen
includes highly immunogenic epitopes that are not specific to
the antigen of interest. This pitfall is common when similar
posttranslational modifications occur among antigens that dif-
fer markedly in amino acid sequence. Unfortunately, the source
and details of antigen purification or the isoforms of the tar-
geted protein recognized by an antibody preparation are seldom
reported for commercial primary antibodies.237 Other factors
affecting immunogenicity include size, conformation, and elec-
trical charge of the antigen; use of adjuvants; and dose and
route of administration of the antigen.
Antibody Structure. Immunoglobulins are ‘‘Y’’ shaped and
consist of 2 identical light chains and 2 identical heavy chains
(Fig. 1). The heavy chains determine the antibody class. The
tail of the Y, called Fc (Fragment, crystallizable), is composed
of 2 heavy chains on the C-terminal side.46 Each end of the
forked portion of the Y is called the Fab (Fragment, antigen
binding) region. The light chains of most vertebrate antibodies
have 2 distinct forms, k and l. In any immunoglobulin mole-
cule, both light chains and both heavy chains are of the same
type. The light chains are divided into the C-terminal half,
which is constant and called CL (Constant: Light chain), and
the N-terminal half, which has abundant sequence variability
and is called the VL (Variable: Light chain) region. The Fab
(antigen-binding) region of the immunoglobulin has variable
and constant segments of the heavy and light chains. The Fc
portion determines biological functions and permits antibody
binding to other antibodies, complement, and immune cells
with Fc receptors. This portion of the Ig is needed for multistep
IHC techniques284 and is largely responsible for nonspecific
background reactivity due to nonimmune adherence of antibo-
dies (Abs) to the tissue section. Background can be diminished
by the use of only Fab or F(ab)2 portions of the Ig molecule for
IHC; however, without the Fc portion, antibody binding to
solid substrates, such as tissues, is less stable. The specific
binding of an antibody to an antigen occurs via hypervariable
regions of both heavy and light chains of the amino termi-
nus.284 The antigen-binding site of an Ab is called the paratope.
Epitopes, usually 5 to 21 amino acids long, are the regions of an
antigen (Ag) that bind to antibodies.141 In an immunologic
reaction, one of the most important criteria for antibody bind-
ing is the tertiary structure of the epitope (ie, the way in which
peptide chains of a protein are folded or interact with adjacent
peptides). The paratope interacts with the tertiary structure of
the epitope through a series of noncovalent bonding (see
below). The more bonding, the greater the affinity and
avidity of the antibody. IgG antibodies are bivalent (have 2
identical arms used in antigen recognition). This is a key com-
ponent of multiple-layer immunohistochemical methods. Other
immunoglobulins, except secretory IgA, which is tetravalent,
and IgM, which is decavalent, are also divalent.
Epitopes are classified as linear or conformational.39 Linear
epitopes are a group of 5 to 7 contiguous amino acids.
Conformational (discontinuous) epitopes, the typical form,
consist of small groups of amino acids brought together by con-
formational folding or binding.39 Although most epitopes
involved in immune responses are believed to be conforma-
tional, data support the concept that antibodies used in FFPE
tissues recognize mainly, or exclusively, linear epitopes.39,347
The Nature of Antigen-Antibody Interactions. From a biochemical
point of view, Ag-Ab interactions are somewhat unusual.284 The
bonds are weak (mostly hydrophobic, van der Waals, and elec-
trostatic) and not covalent. Hydrophobic bonds develop between
macromolecules (interatomic or intermolecular) with surface
tensions lower than that of water. Hydrophobic interactions are
imparted mainly through the side chain amino acids phenylala-
nine, tyrosine, and tryptophan.31 By their lower attraction to
water molecules, these amino acids tend to link with one
another. Electrostatic (Coulombic) interactions (also called
ionic bonds) are caused by attractive forces between 1 or more
ionized sides of the antigen and oppositely charged ions on the
antibody-active site. These typically are the carboxyl and the
amino groups of polar amino acids of the Ag and Ab molecules.
van der Waals forces are weak electrostatic interactions between
dipolar molecules or atoms.2 van der Waals forces and electro-
static attractions are maximal at the shortest distances. There-
fore, precise juxtaposition of oppositely charged ions on
epitopes and paratopes favors strong electrostatic bonding.2
Hydrogen bonds are the result of dipole interactions between
OH and C¼O, NH and C¼O, and NH and OH groups with bind-
ing energy of the same order of magnitude as that of van der
Waals and electrostatic interactions. Their importance in Ag-
Ab interactions is diminished by the necessity of a precise fit
between molecules. Although there are situations with only 1
type of interaction, for most polysaccharide, glycoprotein, and
polypeptide Ags, the Ag-Ab bond is a combination of van der
Waals forces and electrostatic interactions.2 Most protein
antigens are multivalent, and each valency site is generally an
antigenic determinant (epitope) with a completely different con-
figuration from the other sites; a monoclonal antibody (MAb)
can react with only 1 valency site of such an Ag.391 Table 3 lists
the major factors that affect antigen-antibody binding.
The antibody-antigen bond is reversible, and its strength
can be quantified.149 Affinity is a thermodynamic expression
of the binding strength of an antibody (paratope) to an anti-
genic determinant (epitope).213 The affinity constant (Ka) rep-
resents the amount of antibody-antigen complex formed at
equilibrium.
Ab Ag Ab ~ Ag x caloriesaK
Ka ¼½Ab � Ag�½Ag� � ½Ab�
where Ab*Ag are antigen-antibody complexes and the
bracketed terms indicate molar concentrations. Ag-Ab affinity
constants range from below 105 L/mol to 1012 L/mol (an
54 Veterinary Pathology 51(1)
Ag-Ab complex with a Ka of 1012 L/mol has 1000-fold greater
affinity than one with a Ka of 109 L/mol). Affinity can be deter-
mined precisely only for monoclonal antibodies (MAbs);213 for
polyclonal antibodies (PAbs), composed of antibodies with dif-
ferent affinities, affinity can only be estimated. Another
method currently used by antibody manufacturers to calculate
the affinity of antibodies is determining the equilibrium disso-
ciation constant (Kd). Most antibodies have Kd values in low
micromolar (10–6) to nanomolar (10–7 to 10–9); very high affi-
nity antibodies have picomolar (10–12) values. In other words, a
high Ka value (eg, 1012) will correspond with a very low Kd
value (eg, 10–12). New high-throughput technologies such as
microarray-based kinetic constant assays201,202 allow the Kd
calculation for a large number of substances simultaneously.
The affinity of an antibody for an antigen influences the
sensitivity and specificity of an immunologic reaction.149
Intrinsic affinity is determined by the sequence of amino acids
of an epitope, which in turn determines the specificity of an
antibody.149 In general, high-affinity antibodies bind more
antigen in less time than low-affinity antibodies, so the higher
the affinity, the more dilute the antibody solution can be.113,149
Avidity, or functional combined bond affinity, is a measure
of the overall binding intensity between antibodies and a
multivalent antigen.149,213 Avidity is the result of the affinity
or affinities of the antibody for the epitope(s), the number of
antibody binding sites, and the geometry of the antigen-
antibody complexes.213 Thus, an antibody of IgM (decavalent)
has a higher avidity (although typically lower affinity) than an
antibody of IgG (bivalent) type.149,213 High-avidity antibodies
have multiple sites for secondary reagent binding, which is
paramount in IHC.213 The lower the affinity, the longer the reac-
tion time; prolonged incubations may increase the nonspecific
background.149 The functional affinity of a PAb may be higher
than that of a MAb due to its capacity to bind multiple epitopes
on a single antigen.149 To increase the functional affinity of
MAbs, cocktails of antibody clones targeting the same protein
are sometimes used.149
Monoclonal and Polyclonal Antibodies
Polyclonal Antibodies. Polyclonal antibodies are produced by
immunizing rabbits and various other species (mouse, goat,
donkey, horse, hamster, sheep, guinea pig, rat, chicken) with
purified antigen. Chickens transfer abundant IgY (IgG) into
egg yolk, which makes harvesting antibodies from eggs more
convenient than the typical bleeding procedure used in mam-
malian species.46,213 Polyclonal antibodies have higher affinity
and broader reactivity but lower specificity in comparison to
monoclonal antibodies.141 Polyclonal antisera include several
different antibodies to the target protein plus, if not affinity pur-
ified, irrelevant antibodies in concentration up to 10 mg/
ml.89,237 Polyclonal antibodies are more likely than MAbs to
identify multiple isoforms (epitopes) of the target protein.
However, a polyclonal preparation that has not been affinity
purified may be heterogeneous due to the combined presence
of high-affinity antibodies and weakly immunogenic epi-
topes.237 Variations in antibody titer and quality, depending
on the animal immunized, also fluctuate from lot to lot.255 The
‘‘immunologic promiscuity’’ of PAbs, which can amplify anti-
gen detection by recognition of multiple epitopes, can be a dis-
advantage: the greater the number of different antibodies to the
target protein, the greater the likelihood of cross-reactivity with
similar epitopes in other proteins and, therefore, the likelihood
of false positives.237 Many proteins share immunogenic
epitopes. For instance, common immunogenic domains of
fibronectin III are present in at least 65 unrelated proteins.41
Monoclonal Antibodies. Monoclonal antibodies are produced by
the technique of Kohler and Milstein,196 in which an animal,
usually a mouse, is immunized with purified antigen. The
specific antibody-producing B lymphocytes are then harvested
from the spleen and fused with mouse myeloma cells to pro-
duce immortal hybrid cells that produce Igs specific for a single
epitope (MAbs). The immortalized hybrid cells form hybrido-
mas that are selected for the desired specificity and can be
maintained in cell cultures (highly pure antibody but at low
concentration) or in the peritoneum of mice (ascitic fluid with
10- to 100-fold higher Ab concentration than in cell culture, but
with nonspecific proteins and extraneous endogenous Igs). One
advantage of monoclonal over polyclonal antibodies is their
higher specificity (Table 4). In addition, background reactivity
due to nonspecific Igs is reduced (ascites fluid) or nonexistent
(cell culture supernatant). The high specificity does not elimi-
nate the possibility of cross-reactivity with other antigens
because MAbs target epitopes consisting of only a few amino
acids, which can be part of multiple proteins and peptides.254
For instance, an anti–human proinsulin antibody cross-reacts
with both insulin and glucagon-secreting cells.23 Because this
binding is to an identical epitope, the IHC reaction is virtually
indistinguishable from that with the intended epitope.365 It can
be difficult to determine whether immunoreactivity reflects
shared epitopes (cross-reactivity) or epitopes resulting from
protein cross-linking during fixation with aldehydes.141
Table 3. Main Forces Involved in Antigen-Antibody Binding.391
Types of Forces Ag-Ab Binding Favored by Ag-Ab Dissociation Favored by
van der Waals High incubation temperature Reduction of surface tension bufferElectrostatic Neutral pH of buffer Extreme pH of buffer
Low ionic strength of buffers High ionic strength bufferLow incubation temperature High incubation temperature
Ramos-Vara and Miller 55
Rabbit Monoclonal Antibodies. The production of rabbit MAbs
has been facilitated by the development of antibody
libraries.278,283,307 The advantages of rabbit over mouse MAbs
include higher affinity (probably a result of high glycosyla-
tion),149 suitability for use on mouse tissues without special
procedures, increased specificity in some cases, and less need
for antigen retrieval methods.57,132,211,313,417 Nevertheless, in
a study comparing 10 rabbit MAbs with 10 mouse MAbs tar-
geting the same protein in animal tissues, rabbit MAbs were
superior for some markers, but there was no significant overall
improvement in immunoreactivity.396
Protein A and Protein G. These cell-wall components of Staphy-
lococcus aureus (A) and group G (G) streptococcal strains46 are
used in IHC for their high affinity for the Fc portion of IgGs.
The IgG of many species can be bound to protein A or protein
G, creating a versatile label when conjugated to enzymes or
fluorochromes. Protein A/G is a genetically engineered product
of Bacillus sp that combines the IgG-binding domains of
protein A and protein G. Reportedly, it has higher affinity in
various species than protein A or protein G alone.46
Antibodies to Phosphorylated Proteins. These antibodies are spe-
cific for a protein activation state, so may indicate a biological
state rather than the mere presence of a protein.149,222 However,
the production of these antibodies is challenging because the
phosphorylation sites are at consensus sequences shared by
many proteins.149
Mouse-on-Mouse IHC. The use of mouse MAb for IHC of mouse
or rat tissues results in excessive background labeling due to
the binding of the secondary antibody (anti–mouse immuno-
globulins) to endogenous immunoglobulins in the interstitium,
plasma, B lymphocytes, and plasma cells.49,116 This excessive
background labeling has hampered or even precluded IHC with
mouse MAbs in murine tissues; however, commercially avail-
able mouse-specific detection systems have eliminated this
problem. One such system uses blocking steps before and after
adding the primary antibody; another method preincubates the
primary antibody with biotinylated anti–mouse Fab complexes
(used as secondary antibody), thereby blocking free binding
sites in the complexed secondary antibodies with normal
mouse serum before adding the antibody mix to the tissue sec-
tion.116,152,227 Secondary antibodies to mouse IgGs may also
react with Igs of other species producing the same type of non-
specific background in plasma cells, serum, or any tissue con-
taining IgGs. The use of isotype-specific secondary antibodies,
because of the low serum concentration of each isotype (20%–
25%), is a relatively inexpensive method to minimize this type
of background.49
What Makes an Antibody Good for Immunohistochemistry? Speci-
ficity and sensitivity are the most important traits. Specificity is
inherent in the primary antibody. Molecular specificity, the
selectivity of an antibody for a particular epitope of the target
antigen, depends on its molecular structure.149 The diagnostic
specificity of an antibody against infectious agents is quantified
as the proportion of samples from known uninfected animals
that test negative in a given test.419 The diagnostic specificity
of an antibody against cellular/tumor markers is the expected
presence or absence of immunoreactivity in certain cell types,
tissues, or tumors (see Controls in Immunohistochemistry
section).369 Analytical sensitivity is determined by the least
amount of antigen needed to produce a positive reaction. Poly-
clonal antibodies may be more sensitive than MAbs because
they bind different epitopes on a single antigen.369 Diagnostic
sensitivity, the proportion of known positive samples that test
positive in a given test,374,419 is best established by comparing
test results on FFPE tissue with results using another antibody
validated for the same analyte or a non-IHC method, such as
Table 4. Comparison of Polyclonal and Monoclonal Antibodies for Immunohistochemistry.
Polyclonal Ab Monoclonal Ab
Origin Multiple species Mouse, rabbitTotal amount of antibody 5–20 mg/ml 0.05 mg/ml (serum-free medium culture supernatant)
1–10 mg/ml (ascites)Amount of specific antibody 0.05–0.2 mg/ml 0.05 mg/ml (serum-free medium culture supernatant)
0.9–9 mg/ml (ascites)Production Easy and inexpensive More technologically challenging and expensive
Large volume of antibody from the same animal Limitless amount from hybridoma cell linesEpitope recognition Multiple, including multiple isoforms of the same protein One single epitopeSpecificity Low to high HighAffinity Variable (both high and low) HomogeneousAvidity High Lower than polyclonal antibodiesFixation tolerance High Variable to lowFixation effects Multiple SingleMolecular specificity Lower than for monoclonal antibodies High, resulting in less background (background from
mouse antibodies in ascites fluid)Antibody batch heterogeneity Variable Minimal
Limited number of batches Unlimited number of batches
56 Veterinary Pathology 51(1)
culture or polymerase chain reaction (PCR). Nonspecific bind-
ing of antibodies in IHC can be reduced or eliminated with
reagents (including the primary antibody) of good quality;
therefore, the nonspecific binding blocking step included in
most IHC protocols may be unnecessary in some instances.50
Finally, antibody selection depends on its performance in
tissue sections (see standardization and validation below). As
Kalyuzhny181 wrote, ‘‘Having a good antibody for immunohisto-
chemistry is not only about getting a strong staining signal with
low background, but also about knowing the staining makes sense
in terms of its histological and physiological relevance.’’
Presentation of Commercial Antibodies
For a well-characterized antibody, the manufacturer’s package
insert should provide all pertinent information.368 For compa-
nies specialized in antisera production, this should include the
nature of the antibody (eg, purified, whole serum, supernatant,
ascites, immunoglobulin isotype), host (eg, mouse, rabbit) in
which it was produced, protein concentration, immunogen used
(including epitope and molecular weight, if known), species
reactivity/expected reactivity (eg, human, mouse; others not
known), cellular localization (eg, cytoplasmic, membrane,
nuclear), recommended positive tissue controls, applications
(eg, immunoprecipitation, Western blotting, enzyme-linked
immunosorbent assay, immunohistology–formalin/paraffin
and frozen), antigen retrieval, suggested antibody dilution, and
pertinent references. Manufacturers may add client reports on
species cross-reactivities. Many catalogs also include images
of the immunoreaction and Western blot gels with the molecu-
lar weight of the targeted epitope(s) and the percent protein
homology across species. The protein concentration of an anti-
body can be used to estimate the working titer; however, this is
only accurate for MAbs in which the specific antibodies consti-
tute the bulk of the reagent. For PAbs, protein concentration
does not predict performance due to the presence of nonim-
mune proteins or antibodies with variable affinity, in addition
to specific Igs. The manufacturer should be contacted for
additional information on reactivity under certain conditions
(formalin fixation) or species cross-reactivity if not indicated
in the catalog. Data provided by only very few manufacturers
in selected antibodies are their affinity constant/dissociation
constant and the possibility of cross-reactivities with other anti-
gens (eg, serotypes of viruses, other related viruses or bacteria,
cell antigens) that may result from fixation or antigen retrieval
procedures.
How to Find the Antibody That Matches Your Needs. Antibodies
are selected based on consultation with other scientists, publi-
cations, and information from manufacturers.47 Useful Internet
resources include the Human Protein Atlas (http://www.protei-
natlas.org/), which contains information on protein and subcel-
lular profiles of numerous human cancers.25,276,378 The Atlas
lists more than 17 000 antibodies targeting proteins from more
than 14 000 genes with high-resolution images to depict reac-
tivity of the antibodies in tissue sections. Other sites with
information on commercial antibodies are http://www.anti-
bodypedia.com, http://www.biocompare.com, http://antibodyr-
esource.com, http://www.antibodydirectory.com, and http://
www.linscottsdirectory.com. Some provide information about
IHC cross-reactivity and techniques for FFPE tissues or frozen
sections for animal species. The South Dakota State University
database is dedicated to IHC in animal samples (http://
www.sdstate.edu/vs/adrdl/database/). The PHL Murine Immu-
nohistochemistry Database (http://ncifrederick.cancer.gov/rtp/
lasp/phl/immuno/) lists many biomarkers with specific proto-
cols for IHC in mouse tissues.
Detection Systems: The Sensitivity Issue
The primary, secondary, or tertiary antibodies of a detection
system are labeled with reporter molecules (labels) to allow
visualization of the antigen-antibody reaction. Suitable labels
include fluorescent compounds, enzymes, and metals.218,370
The most common labels are enzymes (eg, peroxidase, alkaline
phosphatase, glucose oxidase).303 Enzymes in the presence of
their substrate and a chromogen produce a colored precipitate
at the site of the antigen-antibody reaction.284
The detection system is important to maximize sensitivity of
an IHC test and to optimize visibility of the immune reaction
with the fewest steps and in the shortest time.149 In addition, the
detection system must be reproducible, be accurate, and render
a high signal-to-noise ratio. Other factors to consider in selec-
tion of a detection system include (1) the expertise/experience
of the technician, (2) type of antigens to be detected (less abun-
dant antigens need highly sensitive methods), (3) number of
tests (antibodies) available (different antibodies may require
different detection systems), (4) species/tissue idiosyncrasies
(eg, amount of endogenous biotin), and (5) budget.293 Last but
not least, the detection system must be compatible with the ani-
mal species. A detection system with excellent performance in
human tissues may not perform well in animal tissues. There is
a trend in companies marketing IHC detection systems for
veterinary use to optimize their detection kits based on the spe-
cies evaluated (eg, PromARK for dogs and cats, food animals,
etc), reducing the chances of background or false-positive
labeling. In this article, detection systems are classified as
direct or indirect methods.
Direct Methods. Direct detection methods are a 1-step process
using a primary antibody conjugated (labeled) with reporter
molecules,66 such as fluorochromes, enzymes, colloidal gold,
or biotin.275 The direct method is quick but lacks sufficient sen-
sitivity for the detection of most antigens in routinely processed
tissues (Fig. 11).
Indirect Methods. The need for more sensitive antigen detection
prompted Coons et al67 to develop a 2-step method. The first
layer of antibodies is unlabeled, but the second layer, raised
against the primary antibody, is labeled (Fig. 11).275 The sensi-
tivity is higher than with a direct method because (1) the unla-
beled primary antibody retains full avidity with stronger Ag
Ramos-Vara and Miller 57
Figure 11. Direct and indirect immunohistochemistry (IHC) methods. Figure 12. Avidin-biotin complex (ABC) method. Figure 13. Labeledstreptavidin-biotin (LSAB) method. Figure 14. Peroxidase-antiperoxidase (PAP). Figure 15. Polymer 1-step method. Figure 16. Tyramide-biotin immunoperoxidase method. Figure 17. Nonspecific labeling with inappropriate detection system. Two detection systems were used:LSABþ (a, b), a universal detection system labeling rabbit, mouse, and goat primary antibodies; EnVisionþ (ENVþ) (c, d), a polymer detectionsystem labeling only mouse primary antibodies. Upper panel: Carcinoma, skin; dog. Minimum background with LSABþ (a) and none withEnVisionþ (c). Lower panel: Mammary gland; goat. Extensive background with LSABþ (a) due to binding to endogenous goat immunoglobulins.
58 Veterinary Pathology 51(1)
binding, and (2) the number of labels (eg, peroxidase) per
molecule of primary antibody is higher, increasing the reaction
intensity. Indirect methods can detect smaller amounts of anti-
gen with less primary antibody because at least 2 labeled
immunoglobulins can bind each primary antibody molecule.
Indirect methods are also more convenient than the direct
method because the same secondary antibody can be used to
detect different primary antibodies, provided that the latter are
raised in the same species.275
Avidin-Biotin Methods
Avidin is a large glycoprotein, extracted from egg white,
with 4 binding sites per molecule and high affinity for
biotin. Biotin has 1 binding site for avidin and can be
attached through other sites to an antibody (biotinylated
antibody) or another macromolecule, such as an enzyme,
fluorochrome, or other label.275 The increased sensitivity
of avidin-biotin methods reflects the larger number of biotin
molecules (reporter molecules) that can be attached to a pri-
mary antibody.134,159,160
One of the most common avidin-biotin methods is the
avidin-biotin complex (ABC) method, in which the secondary
antibody is biotinylated, and the third reagent is a complex of
avidin mixed with biotin that is linked with an appropriate label
(eg, enzyme) (Fig. 12). The avidin and labeled biotin are incu-
bated for about 30 minutes before application, resulting in the
formation of a large complex with numerous molecules of
label. The proportion of avidin to labeled biotin in the third
reagent must leave some sites free to bind biotin on the second-
ary antibody.275 Another commonly used avidin-biotin method
is the labeled avidin-biotin (LAB) or labeled streptavidin-
biotin (LSAB) (Fig. 13). This method uses a biotinylated
secondary antibody and a third reagent of peroxidase (or alka-
line phosphatase)–labeled avidin. Its sensitivity is higher than
that of the standard ABC method.90
A disadvantage of any avidin-biotin system is the possibility
of high background. Avidin can produce background by bind-
ing lectins and negatively charged tissue components through
its carbohydrate groups or through electrostatic binding
because its isoelectric point is 10. This background can be
diminished by substituting streptavidin for avidin. Streptavidin,
produced by the bacterium Streptomyces avidinii, has a neutral
isoelectric point, resulting in marked reduction of electrostatic
interactions with tissue. In addition, because streptavidin does
not bind lectins, background labeling is less likely but still
possible due to endogenous biotin. Endogenous biotin activity
or tissue affinity for avidin, known as endogenous avidin-biotin
activity (EABA), is particularly common in tissues that are rich
in biotin, such as the liver, brown fat, adrenal cortex and
kidney. Mast cells also have EABA.138 EABA is accentuated
by HIER but also develops in tissues subjected to other types
of AR.56,187,239,257,275 EABA is typically in the cytoplasm but
has been reported in the nucleus.250,257,342,428 Commercially
available EABA blocking reagents (pure avidin and biotin
solutions) are expensive, so many laboratories use ‘‘home-
made’’ solutions with egg white as a source of avidin and 5%powdered milk as a source of biotin.12,84,175,239,285,418
Non–Avidin-Biotin Methods
Peroxidase-Antiperoxidase (PAP) Method. This indirect method
consists of 3 layers.357 The third layer, which for a rabbit
primary antibody is a rabbit antiperoxidase, is coupled with
peroxidase in proportions to form a stable complex (peroxi-
dase-antiperoxidase) of 2 rabbit IgG molecules with 3 peroxi-
dase molecules, one of which they share (Fig. 14).275 The first
and third layers are bound by a bridge layer of immunoglobulins
(in this example, anti–rabbit Ig). The key is to add the secondary
(bridge) antibody in excess so as to bind the primary antibody
through one binding site and the PAP complex through the other.
The peroxidase-antiperoxidase (PAP) method is more laborious,
but has 100- to 1000-fold higher sensitivity than the 2-step
indirect method. PAP reagents are available for use with goat,
mouse, rabbit, rat, and human primary antibodies.370 Although
PAP IHC was popular before the advent of avidin-biotin meth-
ods, its lower sensitivity limits its use today.
Polymeric Labeling 2-Step Method. This method uses a compact
polymer to which 4 to 70 molecules of enzyme (peroxidase
or alkaline phosphatase) plus 1 to 10 molecules of the second-
ary antibody are attached (Table 5).149,411 Its advantages are
(1) simplicity compared with the 3-step methods, (2) equal
or higher sensitivity than ABC or LSAB methods, and (3)
lack of background due to endogenous biotin or avi-
din.269,318,335,370,398 However, this method is usually more
expensive than ABC or LSAB methods (Fig. 15). Numerous
companies have commercialized polymer-based detection sys-
tems (eg, EnVision, PowerVision, ImmPRESS, MACH 4M),
which are the method of choice in many laboratories. The sen-
sitivity can be increased with a 3-step method, in which a
bridge antibody links the primary antibody and the polymer
complex. Newer detection kits have replaced dextran or other
macromolecules with smaller polymers (micropolymers) to
increase access to the target antigen, resulting in higher sensi-
tivity, lower background, and reduced nonspecific binding.149
Catalyzed Signal Amplification. This method is based on the abil-
ity of tyramide to adhere to a solid substrate (eg, tissue section)
following oxidation/radicalization.131 The procedure, adapted
for IHC,3 is based on the deposition, catalyzed by horseradish
peroxidase (HRP), of biotinylated or FITC-conjugated tyra-
mide at the location of the antigen-antibody reaction. Free
Figure 17. (continued) No background when using EnVisionþ (c). (b, d) Negative reagent controls. Immunoperoxidase-DAB for cytokeratinsusing a mouse monoclonal antibody, hematoxylin counterstain. Figure 18. Double immunolabeling using 2 mouse monoclonal antibodies withsame class isotype immunoglobulin. Figures 11 to 16 and 18 are modified from Ramos-Vara,284 with permission from Veterinary Pathology.
Ramos-Vara and Miller 59
radicals formed during the HRP-tyramide reaction bind cova-
lently to electron-rich amino acids (eg, tyrosine) of nearby pro-
teins.48 The reactive intermediates are so short-lived that
biotinylated tyramide is deposited only at or near its site of pro-
duction.143 The biotin conjugated to the tyramide subsequently
complexes with avidin conjugated to HRP.143 This method is
complex and laborious because it involves an initial avidin-
biotin procedure followed by the tyramide reaction (Fig. 16).
However, its sensitivity is 5- to 10-fold higher than that of the
regular avidin-biotin method;350 others claim an even greater
increase in sensitivity.234 This method is suitable for antigens
present in very low amounts. However, background can be a
problem, particularly when using HIER,140 so prolonged treat-
ment to quench endogenous peroxidase or EABA is usually
necessary. Modifications using fluoresceinated tyramide result
in marked reduction or ablation of background from endogen-
ous biotin.139,140,259,389
Immuno-Rolling Circle Amplification. Rolling circle amplification
(RCA) increases the signal of the immunologic reaction with-
out increasing the noise (background) that can occur with the
tyramide amplification method. Rolling circle amplification
is a 2-part, surface-anchored DNA replication used to visualize
antigens (immunoRCA). The first part is an immunologic
reaction (antigen-antibody binding); the second part is an
isothermal nucleic acid amplification using a circularized
oligonucleotide primer.216,325,436 The primer is coupled to the
antibody, so in the presence of circular DNA, DNA polymer-
ase, and nucleotides, the rolling circle reaction results in a DNA
molecule consisting of multiple copies of the circular DNA
sequence that remain attached to the antibody. The amplified
DNA can be detected by hybridization with labeled comple-
mentary oligonucleotide probes.325 The main differences
between RCA and PCR is that the former can amplify nucleic
acid segments in either linear or geometric kinetics under iso-
thermal conditions, and the product of amplification remains
connected to the target molecule.216,325 The linear mode of
RCA can generate 105-fold signal amplification during a brief
enzymatic reaction. ImmunoRCA is sensitive enough to detect
a single antigen-antibody complex.325
Polyvalent Detection Systems. Many manufacturers offer polyva-
lent (sometimes called universal) detection systems. They
differ from 1-species detection systems in that the secondary
reagent is a cocktail of antibodies against immunoglobulins
from different species, allowing 1 secondary reagent to be used
for both polyclonal (eg, rabbit and goat) and monoclonal (eg,
mouse) antibodies. These systems reduce the complexity of
IHC; however, due to the ‘‘universal’’ recognition by the
polyvalent antibodies, awareness of potential species incom-
patibilities is critical (Fig. 17).
Increasing the Sensitivity of Antigen Detection. The more
sophisticated and highly sensitive detection methods can be
prohibitively expensive. Therefore, standard methods (eg,
PAP, ABC, LSAB) have been modified by combining meth-
ods, repeating steps of the immune reaction, increasing the
incubation time of the primary antibody, or enhancing the
intensity of the chromogen precipitate.§
Detection of Multiple Antigens in a Tissue Section. Multiple
immunolabeling is used to localize different antigens (eg, cell
markers plus infectious organisms) in the same section. The
availability of various detection systems (PAP, ABC,
polymer-based methods, commercial dual-labeling kits),
enzymes, and chromogens with different-colored reactions
makes multiple immunolabeling feasible. A potential pitfall
is false-positive labeling due to cross-reactivity among differ-
ent components of the reaction. This requires careful planning
and the use of multiple controls. Double immunolabeling meth-
ods can be simultaneous (parallel) or sequential.382 Simulta-
neous double immunolabeling is performed with primary
antibodies made in different species; both primary antibodies
are added simultaneously, followed by a mixture of different
enzyme-conjugated secondary antibodies and subsequently
developed with the appropriate chromogen/substrate solu-
tions.149 As a rule, if the primary antibodies are from the same
species, sequential dual labeling is necessary. The risk with
§References 60, 78, 224, 225, 275, 315, 370.
Table 5. Standard Immunohistochemical Protocol Using a 2-StepPolymer-Based Detection System.a
1. Deparaffinize slides and bring them to deionized water2. Antigen retrieval (eg, enzyme, HIER) procedure, if needed3. Rinse in deionized water4. Block endogenous enzyme activities (eg, peroxidase)5. Rinse sections and bring them to buffer6. Nonspecific staining blocking7. Remove excess of blocking solution (do not rinse)8. Incubate section with primary antiserumb
9. Rinse slides with buffer10. Incubate with secondary reagent (immunoglobulin anti–primary
antibody species)11. Rinse slides with buffer12. Incubate slides with tertiary reagent (enzyme-immunoglobulin-
polymer complex)13. Rinse slides with buffer14. Detection of the immune reaction with developing reagents (eg,
DAB and H2O2 for peroxidase)15. Rinse sections with deionized water16. Counterstain with hematoxylinc
17. Dehydrate and coverslipd
HIER, heat-induced antigen retrieval.aThe best incubation times for the primary antibody and other reagentsshould be based on information provided by manufacturers of variousreagents and by personal experience.bIncubation of the primary antibody can be done at room temperature, 37�C,or 4�C. The best incubation temperature should be determined by trial anderror. The incubation chamber should have enough moisture to avoiddessication of the slides.cSelect carefully the type of counterstain based on the chromogen used andlocation of the antigen.dUse water-based coverslipping media when the precipitate of the enzymaticreaction is sensitive to organic solvents.
60 Veterinary Pathology 51(1)
sequential dual labeling is that the second layer of antibodies
(intended for the second antigen) can also bind the first antigen
through the primary antibody (Fig. 18). Therefore, an
intermediate step is inserted between the first and second IHC
reactions to elute the primary antibodies of the first reaction
with potassium permanganate or a solution of glycine-HCl for
several hours or HIER.89,384,387 The use of glycine-HCl with
SDS at pH 2 as an elution agent for sequential dual labeling did
not reduce immunoreactivity.274 The elution step could be
omitted by developing the first reaction with a concentrated
diaminobenzidine solution, which theoretically would block
any residual primary antibody of the first immunoreaction.89
There are commercial kits with an elution or HIER step
between the first and second immunologic reactions.382
Sequential double-labeling techniques are not recommended
if mixed-colored products as a result of colocalization of anti-
gens are expected.382 In other words, sequential dual labeling is
more appropriate for the detection of antigens in 2 different cell
populations or cell compartments (eg, nucleus and cytoplasmic
membrane). A novel IHC procedure can simultaneously
visualize 5 biomarkers within a single tissue section using the
same chromogen (amino-9-ethylcarbazole [AEC]) and anti-
body elution with KMnO4 and H2SO4.122
Multiple immunolabeling requires stringent controls and
careful combination of enzymes and chromogens to achieve the
best color discrimination (contrast) of the IHC reaction. The
optimal order of antigen detection depends on several factors,
including the need for AR. The choice of counterstain depends
mainly on the color of the immune reaction; it should lightly
stain the tissues and differ sufficiently in color from that of the
chromogen precipitate to avoid confusion. The most frequently
used counterstains are hematoxylin, methyl green, and nuclear
fast red.390 In some situations, particularly with nuclear anti-
gens in small amounts, counterstaining is not recommended.284
The Color of the Antigen-Antibody Reaction. The antigen-antibody
reaction is not visible under the microscope unless labeled. The
most common labels are enzymes, particularly peroxidase and
alkaline phosphatase. Each enzyme has specific substrates and
chromogens to produce a colored precipitate. The common
chromogens impart a brown, red, or blue color to the reaction.
The choice of enzyme and chromogen depends on several
factors, such as the reaction intensity, antigen location, pres-
ence or absence of endogenous pigments, or mounting media
used, but often is a matter of personal preference.390 Many
laboratories prefer peroxidase, but alkaline phosphatase (AP)
is also commonly used and is claimed to be more sensitive than
similar immunoperoxidase methods.229,230 For horseradish per-
oxidase, 3,30-diaminobenzidine tetrachloride (DAB) is the
usual chromogen, imparting a brown color that is insoluble in
organic solvents.284 When endogenous peroxidase activity is
high or melanin pigment is prominent, other chromogens or
alkaline phosphatase should be used.370 3-AEC, another
chromogen for peroxidase, produces a red color. However,
coverslips must be mounted with a water-soluble medium
because AEC precipitate will wash out with organic solvents.
4-Chloro-1-naphthol forms a blue precipitate that is also
soluble in organic solvents. For alkaline phosphatase IHC,
5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium
chloride (BCIP/NBT) (blue, permanent media), fast red (red,
aqueous mounting media), and new fuchsin (fuchsia, perma-
nent media) are the usual chromogens.284 Alkaline phosphatase
is recommended for immunocytochemistry.31 Van der Loos385
reviewed the chromogens used in multiplex IHC with photonic
microscope and spectral analysis. Newer chromogens are more
stable and produce a wider range of colors of the final product.
Multispectral Analysis
Selecting the right set of substrates for multiplex IHC can be
difficult, particularly when antigen colocalization causes
mixing of the colored reaction. A novel alternative approach
to photonic multiplex IHC is multispectral analysis, in which
the colors produced during IHC are converted to a spectrum
analyzed by computer.105,384 Multispectral analysis is also
semiquantitative.384
Quantum dots (QDs) are semiconductor nanoparticles that
can be used in multiplex IHC. QDs emit different wavelengths
depending on their size (1–10 nm), shape, and composi-
tion.174,268,270,430 QDs can be linked to antibodies, oligonucleo-
tides, aptamers, and streptavidin for specific binding to tissue
biomarkers430 and are particularly useful to study tumor
heterogeneity.174,214,215 Advantages of QDs in comparison to
standard organic fluorochromes include narrow emission spec-
tra (less interference in multiplex analysis), higher photostabil-
ity (at least 10 hours), and higher fluorescent signal per unit of
light absorbed.98,268,430 In addition, QD IHC is more accurate
and precise at low protein concentration than traditional IHC.61
Refined techniques combining multispectral analysis and
tissue microarrays have made high-throughput biomarker
quantification and colocalization more accurate and less cum-
bersome.102,163,226 These techniques are described in more
detail in another article.219
Manual vs Automated Immunohistochemistry
Laboratories performing IHC on a routine basis benefit from
automated systems. Automated IHC follows the same proce-
dure as manual IHC, but the procedure is programmed into a
computer that controls the steps executed by the autostainer.
Automated IHC saves technician time and typically produces
more consistent results. It has evolved from rudimentary
machines to refined models that can perform the entire proce-
dure, even multiplex IHC, without human intervention.
Selection of the best autostainer depends on the laboratory’s
needs in terms of the number of IHC tests, procedural complex-
ity, and whether the same platform is to be used for other types
of testing (eg, in situ hybridization). Once the needs for IHC are
defined, the choice of platform may be reduced to a few com-
mercially available instruments. This review does not address
the advantages/disadvantages of each automated IHC platform
Ramos-Vara and Miller 61
but focuses instead on the factors to consider before purchasing
an autostainer.
The number of slides that can fit in an autostainer is critical.
Small units accommodate 20 to 30 slides at a time. Additional
units can be connected and controlled by the same computer,
providing flexibility if demand for testing increases. ‘‘Closed’’
systems versus ‘‘open’’ systems is one of the most hotly debated
choices in IHC platforms. With a closed IHC platform, most
reagents and consumables (pipette tips, slide holders, etc) must
be purchased from the platform’s manufacturer. With an open
system, reagents (including glass slides) from other companies
can be used without affecting the quality of the IHC reaction. In
reality, some platforms classified as ‘‘open’’ are only a bit more
open than a closed one. Closed systems are favored by labora-
tories that run numerous IHC tests on human tissues with little
time for tweaking procedures. It is precisely because most IHC
reagents have been developed for use in human tissues that the
closed system has limited appeal in veterinary medicine, except
for certain regulatory tests (eg, prion testing), in which numer-
ous samples are run simultaneously using a procedure validated
for a particular IHC platform. For routine veterinary IHC, an
open system allows the necessary flexibility to perform IHC
in a wide variety of animal species with both commercial and
homemade reagents.
‘‘Online’’ vs ‘‘Offline’’ Antigen Retrieval. Some platforms allow HIER
as a part of the IHC procedure.251 Although this feature provides
greater flexibility, at least with standard AR, these platforms are
more expensive and require higher maintenance that those with-
out ‘‘online’’ HIER. Several platforms are designed for continu-
ous access, meaning that, as some tests are finalized and the
slides are removed from the stainer, a new batch of slides can
be added without stopping the IHC run. This feature is valued
in human clinical laboratories with high demand for rapid test-
ing. Other platforms, especially those with ISH capability, can
use higher incubation temperature (eg, 37�C) to increase reac-
tion speed. Reagent volume may determine the cost of a test;
some platforms require about 100 ml reagent per slide regardless
of the surface area to be covered, whereas others need up to 3
times more reagent. The need for special devices to allow
incubations with smaller reagent volumes should be considered
in the final cost.
Perhaps the most important factor in selecting an autostainer
is the company behind it—in other words, the quality of
technical service and responsiveness of the company to the
laboratory’s needs. The most sophisticated machine is of little
value if the service provided by the manufacturer is suboptimal.
It is uncommon, particularly in veterinary medicine, for the
manufacturer’s software package to satisfy all the needs of a
particular laboratory. The willingness of a manufacturer to cus-
tomize the software to the individual laboratory’s needs is
important in purchasing decisions. Calculation of the cost per
slide produced with an autostainer must include the annual/
semiannual maintenance service fee. As Rodney Miller,
‘‘Although automated immunostainers have improved the qual-
ity of immunostains in many laboratories, the machines are not
perfect, and it is naıve to assume that obtaining an automated
instrument will guarantee quality and reproducibility.’’239
Storage and Handling of Reagents
The shelf-life of IHC reagents varies depending on their nature
and storage conditions. Many primary antibodies and detection
kits can be used beyond the manufacturer’s expiration date
with proper handling and storage.11,323,377 However, such use
must be properly documented.
Diluent Buffer
Antibodies are attracted to the epitopes of most antigens
initially through electrostatic charges and subsequently through
van der Waals and hydrophobic interactions.32 The isoelectric
point (pI) of polyclonal IgG ranges from 6.0 to 9.5, so the
antigen-antibody reaction is relatively stable at pH 6.5 to 8.5
when using whole sera as primary antibody. Although IgG
MAbs have a similar pI range,143 slight fluctuations in pH can
adversely affect antigen binding.2,427 Reportedly,408 MAbs
perform better in phosphate-buffered saline (PBS) than in Tris
buffer. This is puzzling because sodium ions (responsible for
the ionic strength of a buffer) tend to shield negatively charged
epitopes, thereby diminishing the attraction to positively
charged paratopes of the antibody, especially with alkaline buf-
fers.143 Phosphate ions, on the other hand, promote hydropho-
bic binding, which could explain the superiority of PBS in
some instances. Many authors conclude that the best diluent
buffer for MAbs and PAbs is 0.05 to 0.1 M Tris buffer (pH
6.0), but there are exceptions.31,33,429 Tris buffer should also
be used for alkaline phosphatase procedures, because the high
concentration of inorganic phosphate competes for the phos-
phatase.89 Background reactivity due to ionic interactions can
be reduced by increasing the NaCl concentration in the buffer
to 0.3 to 0.5 M;277 however, increased ionic strength can
disrupt the binding of specific but low-affinity antibodies, so
it should be used judiciously.
Assay Standardization
Standardization determines the optimal conditions (eg, incuba-
tion time, incubation temperature, dilutions, pretreatment tech-
niques, controls, buffers, detection system) for an IHC
protocol. In IHC, the goal of standardization is reproducible
and consistent results within each laboratory and comparable
results among laboratories even with different procedures.291
Unfortunately, with the exception of the surveillance program
for prion diseases, neither the protocols nor the primary antibo-
dies are uniform among diagnostic laboratories. This lack of
standardization of IHC tests among veterinary laboratories284
is complicated by the scarcity of high-quality antibodies
designed for the infectious agents and cell markers of
animals.291 Particularly in tumor diagnostics, most veterinary
laboratories use batteries of antibodies developed for use in
human tissue.
62 Veterinary Pathology 51(1)
Even in human medicine, there is little guidance or regula-
tion for standardization of IHC tests, although a consensus on
guidelines for quality assurance for IHC has been published
by the CLSI, formerly the National Committee on Clinical
Laboratory Standards.149 Based on the assumption that most
IHC tests are part of the pathologist’s report and not reported
separately,291 the Food and Drug Administration (FDA) has
classified most IHC reagents and kits as ‘‘Analyte Specific
Reagents,’’ thereby exempting them from premarket notifica-
tion. For antibodies used as ‘‘stand-alone tests’’ in human
pathology, premarket notification, and specific FDA approval
are required. For some stand-alone tests in veterinary IHC
(e.g., detection of persistent bovine viral diarrhea [BVD] virus
infections), premarket notification by regulatory agencies (US
Department of Agriculture [USDA]) is not required. However,
for the transmissible spongiform encephalopathies, in which
IHC is a stand-alone test for the prion protein, the USDA
mandates a standardized procedure, depending on the IHC plat-
form, among laboratories.
Practical Standardization of a New Antibody. Tissue preservation
affects performance of the primary antibody. Frozen sections
typically require less incubation time than FFPE sections and,
depending on the fixative, may not require AR. Although
several commercially available fixatives reportedly preserve
epitopes for IHC better than formalin, they are more expensive
and often less suitable for routine microscopy.291
Primary antibody characterization. The use of a primary anti-
body in a species other than the intended one requires scrutiny
of the specification sheet and additional testing. A Western blot
can be used to demonstrate reactivity with the appropriate
molecular weight antigen in a particular species; however,
Western blot immunoreactivity does not necessarily predict
immunoreactivity in FFPE tissues. Comparison of immunor-
eactivity with that in the ‘‘gold standard’’ species is necessary,
but antigen distribution can vary among species. For viruses,
immunoreactivity should be detected in the appropriate tissues,
cells, and cellular location, as well as in association with
typical lesions. For protozoa and bacteria, consistent morphol-
ogy and location of the labeled organisms supports IHC results.
For cell markers, reactivity should be detected in the appropri-
ate cell or tissue compartment (Fig. 19). More information on
primary antibody evaluation is in the section on test validation.
Antibody dilution. The optimal dilution of the primary anti-
body is that titer with the highest ratio of ‘‘signal’’ (specific
reaction) to ‘‘noise’’ (background labeling) (Fig. 20).149 The
optimal titer should produce appreciable differences in labeling
intensity within and among samples such that strong- and
weak-positive reactions are distinct from the negative
reaction.149 This dynamic range of labeling is particularly
important in scoring immunoreactivity for prognostic or thera-
peutic decisions.149
Primary antibody dilutions for one type of tissue may not be
optimal for another type. The ionic strength of the diluent can
be adjusted to improve the signal-to-noise ratio, particularly for
PAbs in liver sections.431 In a diagnostic setting, however, the
optimal working titer should accommodate a range of relevant
tissues. Tissue processing for IHC must be standardized to
match that used in standardization tests to avoid effects on the
optimal titer.
The manufacturer’s data on antibody performance in FFPE
tissue can be used as a guideline for the starting dilution.
Depending on the antibody type (monoclonal or polyclonal)
and quality, the working concentration should be 1 to 5 mg/ml
and can be reduced with AR procedures.47 Secondary antibo-
dies (working concentration, 5–10 mg/ml47) and detection com-
plexes in commercial kits do not need to be titrated.291
To determine optimal dilution of a new antibody, the fol-
lowing 3 sets of 5 slides each are recommended: 1 set without
AR, 1 set with enzymatic AR, and 1 set with HIER. The first 4
slides in each set have sequential 2-fold dilutions of the primary
antibody (typically, the midrange dilution is that recommended
by the manufacturer); the fifth slide is incubated with the neg-
ative reagent control (Fig. 21).54,287,291,303 A similar approach
is recommended by the College of American Pathologists.157
Incubation conditions. The conditions for incubation of the
primary antibody depend on its affinity, ambient temperature,
section characteristics, and procedure.291 Incubation for most
routine protocols is 30 to 90 minutes at RT. Incubating sections
must be completely covered by an adequate volume of the anti-
body solution to ensure even exposure of the tissue and prevent
drying. Shorter incubation at 37�C to 40�C, longer incubation
at RT, or extended (overnight) incubation at 4�C can optimize
immunolabeling.37 Slides should be incubated in a chamber to
prevent evaporation of the antibody solution.149 For longer
incubations and to reduce evaporation, a container of buffer
or water can be placed within the incubation chamber.149
Many antibody traits affect incubation time. Specificity is
important in selecting the best duration of incubation. Lower
affinity antibodies require longer incubation times, higher con-
centrations, or higher incubation temperature.291 Different lots
of the same antibody may vary in concentration or other char-
acteristics and thus require re-titration.
Interspecies variations in IHC reactions result from subtle
changes in the amino acid sequence of the targeted antigen
(Fig. 22).290 Even with proven interspecies cross-reactivity, the
antibody affinity may be decreased in a different species. Pro-
longed incubation, adjusted AR, and/or increased antibody
concentration may be needed for optimal IHC. Antibody speci-
ficity must be verified in each species tested.291
Choice of detection system. Many commercial detection kits,
some optimized for particular animal species, claim to be the
most sensitive, but any detection kit must be validated in house
before routine use. The secondary and tertiary reagents of
these kits may contain antibodies or other compounds that
could react nonspecifically with tissue antigens, increasing
background or producing false-positive results. This is one jus-
tification for negative controls in IHC (see below in validation).
Typically, a non–biotin-labeled polymer detection system is
Ramos-Vara and Miller 63
Figure 19. Each antigen has a typical anatomic compartment. (a) Carcinoma, mouth; dog. Cytoplasmic pattern for cytokeratins. (b) Lymphoma,lymph node; dog. Cell membrane location of CD20. (c) Histiocytic sarcoma, spleen; dog. Although CD18 is a cell membrane antigen, it may beexpressed in a paranuclear (Golgi) location. Inset: Detail of the reaction. (d) Plasmacytoma, skin; dog. The transcription factor MUM1 isexpressed in plasma cell nuclei. Inset: Detail of reaction. Immunoperoxidase-DAB, hematoxylin counterstain. Figure 20. Optimal titrationof the primary antibody (Ab). Lymph node; dog. Prox-1 is a transcription factor of lymphatic endothelial cells. (a) Excess Ab (1/100 dilution)
64 Veterinary Pathology 51(1)
recommended if HIER is used to avoid background from
EABA.54
Postanalytical Phase of IHC
Assay Validation
Validation is a process to affirm the suitability of an assay, once
it has been developed, optimized, and standardized, for a spe-
cific purpose.419 Validation appraises the analytical and diag-
nostic performance of a test.419 A validated IHC test should
be reproducible with high sensitivity and specificity.71 Valida-
tion entails several steps starting with evaluation of analytical
characteristics: specificity and sensitivity, repeatability, and
preliminary reproducibility. 419 Next, diagnostic characteris-
tics, particularly diagnostic specificity and sensitivity, and cut-
off value are evaluated. Finally, the test’s reproducibility and
ruggedness are evaluated with implementation among labora-
tories and continuous monitoring of validation criteria.419 Test
validation requires proof of antibody specificity in an estab-
lished assay. For most IHC tests, this involves detecting any
cross-reactivity of the antibody with unrelated antigens (or the
presence of antigen in a cell or tissue not previously known to
harbor it) or among different tissues species. Moreover, valida-
tion examines the variables that affect the IHC reaction, such as
fixation time and storage of paraffin blocks or unused tissue
sections.291 A single assay may be validated for several pur-
poses by optimizing for each intended purpose.419 For exam-
ple, if an IHC marker (analyte) is to be used to demonstrate
the same antigen in different animal species, antibody dilution,
incubation time, AR, and other test parameters may need
adjustment. An IHC test can be validated by comparing results
among laboratories using similar techniques.16 When possible,
validation compares the IHC test sensitivity to that of the ‘‘gold
standard’’ method for the antigen in question. It may also com-
pare staining patterns and sensitivity with other antibodies tar-
geting the same antigen. Test validation follows test
standardization.419 The CLSI evaluation protocol for a qualita-
tive diagnostic test, such as IHC, in situ hybridization and
Western blotting analysis of 50 positive and 50 negative speci-
mens if validation data are not available from other sources (eg,
antibody manufacturer).71 This target can be difficult to
achieve in veterinary medicine where validation may have to
be an ongoing process. The in-house validation procedure
depends on the antibody and the laboratory. A validated IHC
assay should produce consistent results, regardless of the
method or the laboratory where it is performed.291
As mentioned earlier, validation of an IHC test may require
an additional method (eg, the gold standard) to prove the pres-
ence or absence of the antigen in question. In some cases, the
gold standard method does not prove the presence of a protein
but demonstrates a physiological activity closely associated
with the given protein. An example is the validation by Marti-
net et al228 of an assay to demonstrate autophagy-related pro-
teins. The gold standard to detect autophagy is transmission
electron microscopy (TEM). However, because this method
is time-consuming and not routine, the authors developed an
IHC assay. Autophagy was induced in mice by starvation (pos-
itive control, confirmed by TEM); the negative control was tis-
sue from transgenic mice with the essential autophagy gene
Atg7 deleted. These mice lacked autophagolysosomes by TEM,
even when starved. Different commercial primary antibodies,
putatively specific for autophagy, were used as well as different
fixation procedures, buffers, and AR protocols. The use of Atg7
knockout mice as a negative control proved that some of the
tested antibodies indeed recognized a different protein in
normal and starved mice.
The use of acetone- or ethanol-fixed frozen tissue sections
has been advocated as the gold standard against which IHC
on FFPE should be compared. However, in some instances, fro-
zen sections fixed in coagulating fixatives are suboptimal for
IHC compared with FFPE tissue sections.338 This is particu-
larly relevant when testing low-molecular-weight proteins and
lipoproteins, which are readily extracted by coagulating fixa-
tives.203 The same conclusion was reached by van der Loos383
in studying the distribution of interferon (IFN) g–producing
cells using different fixatives (Fig. 6). This point is corrobo-
rated by international governing organizations of diagnostic
assays, which indicate that the matrix (eg, tissue section) in
validation studies should be identical or closely resemble the
samples to be tested.419
Primary Antibody Characterization. Validation of an assay
requires confirmation that the primary antibody is specific,
selective, and reproducible for its intended use.40 For cellular
markers, a good starting point is Western blot (WB) to demon-
strate reactivity with the appropriate molecular weight antigen
in the tissue and species of interest;320,386 multiple bands or
bands at inappropriate molecular weight could indicate post-
translational modifications or low antibody specificity.40,200,321
results Figure 20. (continued) in muddy nuclear labeling in both endothelial cells and lymphocytes with background in plasma (v). (b) Specificnuclear labeling of Prox-1 in lymphatic endothelium at 1/400 Ab titer. (c) Labeling of lymphatic endothelial cells (arrowheads) with lack of back-ground in lymphocytes or plasma (v) at 1/800 titer. Note hemosiderin in macrophages at bottom (asterisk). Immunoperoxidase-DAB, hema-toxylin counterstain. Figure 21. Antibody standardization for immunohistochemistry (IHC). Three sets of sections (5 slides per set) areused. Two-fold dilutions (1/25 through 1/200) were made (first 4 slides on each row; the last slide is the negative reagent control). Sectionsin each set were treated with no antigen retrieval (AR) (a), enzyme digestion (proteinase K [PK]) (b), or heat-induced epitope retrieval (HIER)with citrate buffer, pH 6.0 (c). Immunoperoxidase-DAB, hematoxylin counterstain. Figure 22. Oral mucosa; horse. IHC with monoclonal anti-bodies (MAbs) to vimentin. (a) Mouse MAb reacted appropriately with mesenchymal cells such as lymphocytes (arrowheads), smooth musclecells of vessels (asterisk), and interstitial cells (solid circle) and did not label epithelial cells. (b) Rabbit MAb had no reactivity. Immunoperoxidase-DAB, hematoxylin counterstain.
Ramos-Vara and Miller 65
Nevertheless, WB immunoreactivity does not necessarily
predict immunoreactivity in FFPE tissues; WB requires antigen
denaturation (therefore, modification of its secondary and ter-
tiary structure) as opposed to the in situ antigen modification
in formalin-fixed tissues, with cross-linking rather than dena-
turation.55,413 Some antibodies bind only denatured protein
immunoblots; others bind only native proteins.55,69 In some
cases, as with HIER, the antigen denaturation by WB may not
be problematic and does not disqualify this procedure, because
IHC detection of most epitopes requires only an intact primary
(linear) protein structure.39,347
Incubating the antibody with excessive blocking peptides
before the IHC reaction (the so-called preadsorption test) has
been used in validation.40,155 However, blocking peptide proce-
dures do not prove antibody specificity or provide information
regarding signal-to-noise ratios,360 and the amount of antigen
that constitutes an ‘‘excess’’ can only be calculated by knowing
the Kd of the antibody.321 Diluting the antibody to the point that
labeling occurs only in expected locations is a more logical
approach.321
Despite their shortcomings, WB and preadsorption tests are
the most widely used methods to determine the specificity of a
primary antibody in a diagnostic setting. Other methods that
require more sophisticated technology and laboratory capabil-
ities include (1) use of knockout animal tissues to demonstrate
the lack of detection of the protein genetically removed from
that animal and (2) use of a transfected cell line expressing the
antigen for the primary antibody.55,320 Comparison of immu-
noreactivity with the ‘‘gold standard’’ species is another option;
however, antigen distribution may vary among species.291 In
the end, antibody specificity is best documented by the appro-
priate use of controls (see below).40
A special situation arises with commercial purified affinity
MAbs that claim specific antigen targeting as demonstrated by
WB on a limited set of tissues or cell culture extracts. By def-
inition, these antibodies are specific for a particular epitope. In
reality, some of these ‘‘commercially validated’’ antibodies
cross-react with unrelated antigens associated with degeneracy
and immune mimicry.63,279 This unexpected cross-reactivity is
difficult to detect without extensive antibody validation with
different tissues by WB or paraffin sections.386 For most
laboratories, this approach is neither practical nor economically
feasible.
A common practice in the biopharmaceutical industry is tis-
sue cross-reactivity (TCR) studies to identify off-target binding
of primary antibodies or antibody-like molecules with a
complementary-determining region; TCR is also used to detect
previously unidentified sites of on-target binding.204 These
pharmaceutical compounds, intended as vehicles of biologi-
cally active molecules, are incubated with microarrays of
human or animal tissues (frozen or, less commonly, fixed);
TCR studies supplement but do not replace in vivo studies.204
In some laboratories, the same MAb (same clone and
antibody presentation) from 2 different companies is used in
the same IHC procedure for validation purposes. We have
experienced aberrant immunoreactivity using an antibody to
CK8/18 from one company, whereas the same clone from a dif-
ferent company produced the expected results (Figs. 23, 24).
This discrepancy suggests that nonactive ingredients of the
monoclonal preparation could have interfered with the immu-
nologic reaction.
Cross-Reactivity. Cross-reaction can result in a false-positive
reaction.419 However, in IHC, 2 main types of cross-reaction,
neither of which necessarily implies a false-positive reaction,
can be used to the diagnostician’s advantage within the proper
context: antigen cross-reactivity and species cross-reactivity. It
is assumed that test conditions are those used during
standardization.291
Antigen cross-reactivity. The structure and amino acid
sequence of an antigen affects its cross-reactivity. A literature
search may reveal reports of antigen cross-reactivity. Lack of
cross-reactivity in one species does not preclude it in
another.291 Molecular mimicry (eg, cross-reactions of infec-
tious agent antigens with normal tissue antigens) may also
occur.353
Evaluation of cross-reactivity in the diagnostic setting
depends on the category of the antibody (against infectious
agents vs cell markers).291 Antibodies against bacteria, proto-
zoa, or fungi should be tested against other microorganisms
that are morphologically similar, belong to the same group,
or produce similar lesions in the organ of interest. Antibodies
to viruses should be tested against other viruses in the same
group that affect the same tissue or produce similar lesions.
Some lack in specificity does not preclude the use of an
antibody, provided it is documented and interpreted. For exam-
ple, an antiserum against porcine coronavirus is used to detect
feline enteric coronavirus, feline infectious peritonitis virus, or
mink and ferret coronaviruses. Some MAbs against infectious
agents cross-react with cellular organelles.
For neoplasms, antibodies to specific cell types or compo-
nents should be evaluated in 1 or more of the following ways:
(1) comparison of immunoreactivity in primary versus second-
ary (metastatic) tumors (Fig. 25),288,297 (2) determination of
immunoreactivity in other neoplasms in the differential diagno-
sis,289,294,296,297,299,302 (3) comparison of immunoreactivity
between nonneoplastic and neoplastic tissues (antigen expres-
sion may be lost or expressed de novo in neoplastic cells)301
or between benign and malignant phenotypes,392 (4) evaluation
of immunoreactivity in tumors with different histologic
patterns or cellular phenotypes,288,295,297 (5) determination of
antibody cross-reactivity between neoplastic and nonneoplastic
cells in the organ of interest (eg, hepatocellular tumors vs sec-
ondary hepatic tumors),298 (6) comparison of reactivity
between wild-type and mutated antigens (eg, p53), and
(7) semiquantitative evaluation of immunoreactivity of various
antibodies targeting the same cell or cell product in various
tumors or tissues to determine their relative diagnostic utility
(Fig. 26).290,301,302
Species cross-reactivity. Although some antigens can be
detected under similar conditions in different species, it should
66 Veterinary Pathology 51(1)
Figure 23. Manufacturer differences in MAbs. Intestine; dog. MAb 5D3 cell culture supernatant for cytokeratins (CK) 8/18. Compare with Fig.24, from a different manufacturer. (a) Expected reactivity in mucosal epithelial cells (solid circle) with no labeling of mesenchymal cells in thesubmucosa (s) or muscularis (m). (b) Detail of the mucosa. (c) Detail of the submucosa and muscularis. Immunoperoxidase-DAB, hematoxylincounterstain. Figure 24. Manufacturer differences in MAbs. Intestine; dog. MAb 5D3 cell culture supernatant for CK 8/18. Compare with Fig.23, from a different manufacturer. (a) Intense labeling of mucosal epithelial cells as well as many mesenchymal cells, including lamina proprialleukocytes (solid circle), fibroblasts, vessel walls, and nerves in submucosa (s) and muscularis (m). (b) Detail of lamina propria immunoreactivity.(c) Detail of submucosal immunoreactivity. Immunoperoxidase-DAB, hematoxylin counterstain. Figure 25. Antigen cross-reactivity. Intestine;dog. The B-lymphocyte marker CD79a is strongly expressed in normal smooth muscle of the submucosa (s) and muscularis (m).Immunoperoxidase-DAB, hematoxylin counterstain. Figure 26. Antibody interspecies cross-reactivity. Skin; horse. (a) Melanocyte markerPNL2 labels neoplastic cells of equine melanoma. (b) Melanocyte marker Melan A is not expressed in equine melanoma. Insets: Immunohisto-chemistry detail. Immunoperoxidase-DAB, hematoxylin counterstain. Figure 27. Infectious bovine rhinotracheitis virus (IBR) infection, colon;calf. Necrotic foci in lamina propia (l) and submucosa (s and arrowheads) have strong IBR antigen expression. Inset: Epithelial cells havenonspecific supranuclear expression in the Golgi zone. Immunoperoxidase-DAB, hematoxylin counterstain. Figure 28. Inadequate heat-induced epitope retrieval (HIER). Liver; horse. (a) Leakage of solution during antigen retrieval left the upper third of the tissue section unex-posed to HIER solution, therefore unable to react with the primary Ab. (b, c) Reactivity was minimal in the portion of the section not exposed toHIER solution (solid circle). Immunoperoxidase-DAB for equine herpesvirus 1, hematoxylin counterstain.
Ramos-Vara and Miller 67
be assumed that antigen detection varies among species.301
Identical antibody clones differ in reactivity among species.
Not all antibodies targeting particular antigens will be reactive
in all species. For example, Melan A labels melanocytes in
dogs, cats, and other species but not in horses.290 It is advisable
to restandardize (optimize) an IHC test for each new 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.291 A diagnostic IHC report should indicate the
degree of confidence in the results based on experience and
publications. The use of tissue arrays from different animal
species permits rapid screening for interspecies cross-
reactivity.173,265,284
Effects of Fixation, Postfixation Treatments, and Equipment onImmunoreactivity. These laboratory variables affect assay devel-
opment and validation.419 Different fixatives have different
effects on immunoreactivity. For each fixative, full standardi-
zation of a test must be done, including incubation conditions,
dilution of the primary antibody, and AR method (see section
on standardization). Standardization is usually done on tissues
with known fixation time (eg, 1–2 days).
Fixation kinetics studies are recommended for each antigen
or epitope as part of the validation process to determine the
assay robustness, defined as its capacity to produce reproduci-
ble results despite small variations in method parameters (eg,
incubation temperature, pH of buffers, batch of reagents, shelf
lives and storage conditions, fixation length).419 A positive
tissue control should be fixed for different durations (eg, 1, 2,
4, 7, 10, 14, 21, or 20 days) and tested under identical condi-
tions to determine the effects of fixation time on reactivity
(ie, intensity and number of cells or organisms detected
(Fig. 3).288,297,403,404
Automated stainers are designed to duplicate manual
procedures and ensure uniform application of each step of the
process. Thus, the use of automated equipment creates a uniform
and standardized testing environment, which will improve intra-
laboratory run-to-run consistency.251,369,433 When results among
different laboratories diverge considerably, technical differences
should be considered as a possible cause.
What Constitutes a Positive/Significant Result? There is no single
answer to this question. IHC assays detect analytes (infectious
agents or tumor marker antigens) in tissues, but diagnosis
requires interpretation of IHC results in the context of clinical,
pathologic, or laboratory findings (the so-called class I IHC
tests).375 For an infectious agent, detection of even 1 organism
indicates infection, but it is another matter to prove that the
agent caused disease. For neoplasms, interpretation is even
more complex, because tumors are often heterogeneous, and
tumor stem cells can differentiate in various directions. The
reported percentage of positive cells required to confirm tissue
origin of a tumor varies considerably. A cutoff value of 10%positive cells has been suggested to classify an IHC test as
positive,375 but the cutoff value is by no means set in stone.136
Some pathologists prefer the words detected or not detected
rather than positive or negative, which underscores the need for
subjective interpretation of the IHC reaction in the context of
the disease.326 Each antigen has a ‘‘different weight’’ in the
final interpretation. A more pragmatic approach would be to set
a cutoff value for each antibody, optimized for a given entity.
For example, the cutoff value for CD34, consistently expressed
in many different mesenchymal cells, should be higher than
that for uroplakin III, which, although highly specific for
urothelial tumors, may be expressed in only a small proportion
of the neoplastic cells.297 For prognostic markers or those used
to customize therapy, a standardized scoring system applicable
to different platforms is required for meaningful results.380,402
How to Distinguish True-Positive From False-Positive Immunoreactiv-ity. The pattern of immunoreactivity, particularly with neo-
plasms, is important in distinguishing a true-positive reaction,
which has some degree of cell-to-cell heterogeneity, from a
false-positive reaction, which lacks heterogeneity, even in the
absence of stromal background reactivity.239 Reactivity of a
biomarker in tissues/cells not reported previously is not
unusual; less commonly, an antigen is detected in a location not
consistent with its function. For example, thyroid transcription
factor 1 (TTF1) expression is expected in the nucleus, but not
cytoplasm, of thyroid and pulmonary neoplasms;299,300 how-
ever, cytoplasmic TTF1 expression has been reported in human
liver neoplasms and confirmed in mitochondria by other meth-
ods.58,267,410 Whether the detected antigen is part of the TTF1
molecule or a cross-reacting antigen is not clear.
Clinical Validation. Clinical validation is based on whether a test
result correlates with case outcome or response to therapy.126
Clinical validation is common in human oncology but requires
access to hospital medical records from numerous cases. In
veterinary medicine, prognostic or therapeutic assumptions are
often based on published information.24,191,319,373,395
Controls in Immunohistochemistry
Positive Tissue Control. A positive tissue control is a tissue known
to contain the targeted antigen detectable by identical IHC
methods to those used in diagnostic cases.149 Positive tissue
controls assess the performance of the primary antibody.149
Fresh-fixed surgical biopsy specimens are preferred over
postmortem specimens, because extended warm ischemia and
autolysis affect immunoreactivity.149 For laboratory animals,
autolysis is less of an issue, and postmortem specimens are rou-
tinely used. Positive tissue controls must be fixed, processed,
and stained in the same way as the test tissue for each antibody
and procedure.306,369 Control tissues should be from the same
species as the test tissue, with few exceptions (see below). The
antigen in the positive control should not be abundant and
should have some heterogeneity in intensity throughout the
sample; weakly immunoreactive areas can be used to detect
subtle changes in antibody sensitivity.369 A positive control
68 Veterinary Pathology 51(1)
with abundant antigen may reduce the chances of identifying
weak-positive cases.149 The presence of the antigen in the
tissue control should be confirmed by another method (eg,
PCR, virus isolation) or documented by publication. For most
laboratories, control tissues are generally obtained ‘‘in house’’
because fixation and processing procedures are those used with
the test tissue. Control tissues from another laboratory or a
commercial source could have been fixed or processed differ-
ently. When possible, the positive tissue control should be on
the same slide as the test tissue to minimize technical variation.
Multitissue blocks and cell lines. Various methods for the
simultaneous study of multiple FFPE tissues in a single histo-
logic section have been reported. In the sausage technique,17
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.
Substitution of placenta as the ‘‘wrapping’’ tissue115 and other
modifications239 to the original method have been reported.
Paraffin-embedded tissue microarrays. These have been used as
controls in human IHC laboratories, mainly for tumor diagno-
sis.158,265,266,284 Validation studies should be carried out on
multitissue control blocks containing both known-positive and
known-negative normal and neoplastic tissues.369 In veterinary
medicine, species-specific tissue microarrays are not commer-
cially available. Species-specific tumor cell lines can be used
for IHC controls (eg, for HER-2/neu);306,310 this approach
provides an identical control for comparison of results among
laboratories.291
Peptide controls. The basis behind peptide controls is that
formalin-fixed peptide epitopes, covalently attached to glass
slides, behave immunochemically like the native protein in tissue
sections.38 Mass production of peptide controls is cheaper than
tissue or cell line controls, creates an identical control for numer-
ous assays, and facilitates standardization of IHC protocols.
Reference control tissues for infectious diseases. The presence or
absence of an infectious agent in a tissue should be assessed by
known IHC reactivity patterns plus another (non-IHC) diagnostic
method.15 For example, Listeria monocytogenes control tissue
could be assessed by IHC (immunoreactivity of extracellular or
phagocytized bacilli) and by bacterial culture. Similarly, BVD
virus control tissue could be assessed by IHC (immunoreactivity
in cytoplasm of infected cells) and by virus isolation or PCR. In
addition, the presence of characteristic lesions of a particular dis-
ease supports use as a standard reference control.291
IHC protocols for infectious diseases should be species
matched with test specimens; alternatively, tissues from other
species infected with the same agent can be used (eg, BVD virus
identification in test tissue from cervids using a bovine control tis-
sue). Nonspecific binding of primary antibody (species-specific
molecular mimicry) or secondary (linker) reagents can occur
among species. An example is the use of goat PAbs against Neos-
pora caninum on goat tissue. The anti–goat IgG secondary
reagent can bind extensively to endogenous goat IgG in the tissue,
resulting in background ‘‘noise’’ that obscures any immunoreac-
tive tachyzoites.291
Reference controls for neoplastic diseases. Often, the only way
to verify the presence of an antigen is by histology, which is not
always accurate. Moreover, a negative result for tumor markers
does not rule out a particular neoplasm because the marker of
interest may not be expressed in a poorly differentiated cell
population. Ideally, controls for tumor cell markers should
consist of the neoplasm of interest, one with heterogeneous
reactivity.369 Control tissues for IHC tumor markers should
also be species matched with test specimens because the detec-
tion of an antigen in a particular tumor and animal species does
not guarantee similar results in a different species.291
Negative Tissue Control. A negative tissue control is known not
to contain the antigen of interest.149,369 A negative control
should be included in each IHC run to verify the specificity
of the primary antibody and the presence or absence of back-
ground (false-positive) reactivity; if false-positive reactivity
(tissue labeling with a pattern similar or identical to that in the
positive control) occurs in the negative control, the test should
be considered invalid.149 At least one ancillary test (eg, PCR,
virus isolation) on the same animal should be used to rule out
the presence of the antigen of interest. As for the positive
control, a negative tissue control should be processed in the
same manner as the case material. Similarly, when using multi-
tissue blocks for antibody validation studies or as tissue con-
trols, the specimens must be fixed and processed as for the
case material.369 In practice, only 1 tissue control is used due
to the common presence of both positive and negative cells
within the positive control. If possible, the positive tissue con-
trol section should be on the same slide bearing the diagnostic
(test) tissue section.291
The lack of labeling with a negative tissue control does not
necessarily validate labeling in the positive tissue control. An
example is observed with some MAbs produced in ascites fluid
labeling the Golgi zone of different types of epithelial cells in
different species (Fig. 27).351 This nonspecific labeling is more
related to the immunized mouse rather than to the type of
immunogen used or the immunoglobulin class (eg, IgG1 or
IgG3) produced in the ascites fluid and has been blocked by
adsorbing the ascites fluid with blood group A1 human erythro-
cytes.351 We have observed similar nonspecific reactivity with
some MAbs. In the end, interpretation of specificity is based on
the expected location of the antigen in a particular cell or cell
compartment.400
Internal Positive Tissue Controls. Internal positive tissue controls
(eg, smooth muscle markers or vimentin in blood vessels) are
present in many test tissues. Labeling in these areas indicates
appropriate immunoreactivity. In addition, with internal
controls, there is no variation in fixation or processing.32,239
Internal controls can be used to assess sample quality by
identifying ‘‘housekeeping’’ or structural proteins present in
relatively constant amounts in certain cell types in a wide vari-
ety of tissues, such as endothelial cells or lymphocytes.369
Ramos-Vara and Miller 69
Reagent (Antibody) Controls. Negative reagent controls (without
the primary antibody) are used to confirm test specificity and
to assess the degree of nonspecific background caused by the
secondary or tertiary antibody.149 Commonly, the primary anti-
body is replaced by 1 of the following: (1) antibody diluent,
(2) same-species nonimmune immunoglobulin at the same con-
centration and in the same diluent, (3) an irrelevant antibody, or
(4) buffer.306,369 Only reagents 2 and 3 qualify as true-negative
controls.291 Ready-to-use, universal negative reagents are com-
mercially available for rabbit and mouse primary antibodies.
With the different quality standards among laboratories, an
agreement on which reagent control is best may not be forth-
coming.400 The current CLIS-approved guideline for IHC also
accepts, as an inferior option, simple omission of the primary
antibody from the protocol.149
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 primary antibodies itself can
serve as a set of irrelevant reagent controls; thus, the need for
multiple negative controls is eliminated. With this method, sep-
arate controls should still be run for each different protocol (eg,
different AR or detection system).369
Quality Assurance/Quality Control
Human IHC laboratories must meet federally mandated stan-
dards of operation as defined by the Clinical Laboratory
Improvement Amendments (CLIA).101 Veterinary laboratories
accredited by the American Association of Veterinary Labora-
tory Diagnosticians (AAVLD) must meet the ISO/IEC 17025
standards for all types of assays, including IHC. This document
establishes the general requirements for testing, including
laboratory organization, document control, test calibration, and
technical requirements for performing a test. Technical require-
ments include personnel training and qualifications, environ-
mental conditions, calibration and validation, equipment, and
reporting.168 In nonaccredited laboratories, diagnostic tests
should be validated by documentation of internal or interla-
boratory performance using reference standards and relevant
diagnostic specimens. This should be corroborated by the
endorsement of organizations, such as the World Health Orga-
nization, by publication in peer-reviewed journals or by direct
comparison with an established method. Following are general
guidelines for quality control (QC)/quality assurance (QA)
standards for veterinary laboratories. More detailed informa-
tion has been published.291
Daily Quality Assurance/Quality Control. Each laboratory should
establish standard operating procedures (SOPs) for histology.
These include schedules for cleaning, maintenance, and moni-
toring logs, along with a daily check of equipment such as
microwave, oven temperature for drying slides, temperature
of water baths, pH meter, reagent containers, and so on (Fig.
28).217 Buffers, antibody dilutions, and other reagents should
be prepared according to standard or manufacturers’ recom-
mended procedures.
Internal Quality Assurance/Quality Control. A biannual internal QA/
QC check of IHC slide interpretation by pathologists is
recommended.
External Quality Assurance/Quality Control. The goal for interla-
boratory comparisons is to document variations in reactivity
among laboratories and to set minimum QA/QC standards for
IHC protocols to be shared by all laboratories.409 Despite its
difficulty, interlaboratory standardization of IHC tests on ani-
mal tissues in veterinary laboratories is in progress. Even in
human medicine, only a handful of IHC tests (eg, Hercep test)
have been validated in such a way that different laboratories
can perform tests with consistent results and not without
controversy.28,156,314 As Fletcher and Fletcher104 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.’’
There are 2 main approaches to interlaboratory
standardization.291
Technical equivalency testing of an IHC test. Unstained slides are
distributed among different laboratories for performance of the
IHC test. Technical equivalency testing evaluates the quality and
interlaboratory consistency of IHC and focuses not only on results
but also on the technical quality of the IHC preparation and arti-
facts that might influence the test results (eg, background staining,
loss of tissue due to harsh treatment). Currently, the Pathology
Committee for the AAVLD Program for Interlaboratory Compar-
ison of Infectious Agent Immunohistochemical Assays recom-
mends an annual, voluntary, immunohistochemical proficiency
test managed by the Pathobiology Section of the National Veter-
inary Laboratory Services, USDA (Ames, IA). Unstained paraffin
slides are sent to participating laboratories, which perform the
required IHC test and interpret the results. Evaluation includes
quality of the IHC procedure and its interpretation. These interla-
boratory tests are common in human medicine and have exposed
differences in IHC proficiency among laboratories that could
affect therapeutic decisions.68,136,309,380,416,426 For ‘‘stand-alone’’
IHC tests in veterinary medicine (eg, scrapie, chronic wasting dis-
ease), participating laboratories must pass an annual proficiency
test.291 Similar requirements have been established for various
biomarkers in human tissues.416,426 External quality assurance for
immunocytochemical preparations is limited.188
Diagnostic equivalency testing using IHC testing as an adjunct. In
this case, both the pathologist’s proficiency and the technical
quality of IHC are evaluated. A brief history and description
of the tissue examined is included with tissue sections. The
pathologist must base the diagnosis on routine (eg, hematoxylin
and eosin) staining and support it with appropriate IHC testing.
Results could be reviewed by a committee to address any
differences among participating laboratories.407
Immunohistochemistry Reporting and Interpretation
If not included in the standard report, the IHC report should con-
tain demographic information pertinent to the case, the tissue
70 Veterinary Pathology 51(1)
that was tested, and the antibody used or the targeted antigen.
Other IHC test details should be filed in the laboratory. Results for
infectious antigens should be reported as ‘‘detected’’ or ‘‘not
detected.’’ The interpretation for infectious disease IHC/immuno-
cytochemistry (ICC) tests should include a disclaimer that any
lack of detectable antigen could be due to the absence of the
antigen in the section or to technical aspects, such as prolonged
fixation or method sensitivity.291 For tumor markers, a descrip-
tion should include the cellular location (eg, cytoplasmic, cell
membrane, nuclear) (Table 6), distribution through the tissue sec-
tion, labeling intensity, and percent positive cells, along with an
interpretation of the test results.149 If antigen location is
atypical, or if testing of a marker has not been validated in the spe-
cies of interest, this should be stated in the report.291 A scoring
system for IHC has been proposed by CLIA (Tables 7, 8).149 Per-
sonnel who read IHC slides should be familiar with the antibodies
used and their specificity and sensitivity, and an experienced
pathologist should review daily IHC preparations for QA/QC.291
Most important, interpretation of IHC results requires famil-
iarity with the expected pattern of immunoreactivity based on
location of the antigen in the cell of interest.425 For example,
labeling of cytokeratins and vimentin should be cytoplasmic;
labeling of TTF1 in lung or thyroid tumors is nuclear.299,300
The degree of specific immunoreactivity varies among cells
and, in some cases, among different cell compartments. Cellu-
lar distribution of some antigens has prognostic significance in
certain tumors.191 Finally, interpretation of an IHC reaction is
only as good as the controls.413
Particularly in leukocytic populations, IHC expression of a
marker can confirm cell type, without documenting the neo-
plastic nature of the proliferation. Molecular demonstration
of clonality supports a diagnosis of neoplasia, for example, in
feline intestinal lymphoma, in which histologic distinction
between neoplastic lymphocytes and reactive lymphocytes of
inflammatory bowel disease is difficult.185,190,246
Patterns of Specific Labeling. Antigens can be present in various
organelles and intracellular locations or outside cells (Fig. 19).
In the nucleus, viral inclusions, inclusions of known or unknown
cause, nucleoli, chromatin, the nucleoplasm, and the nuclear
membrane may be labeled. Other intracellular sites of antigen
expression include mitochondria, lysosomes, rough and smooth
endoplasmic reticulum, lipid droplets, peroxisomes, pigment
Table 6. Staining Patterns and Manual Interpretive Scoring Criteria.
Cellular Location Staining Pattern Scoring Criteria
Nuclear only Staining limited to nucleus Positive/negativePercent positiveIntensity
Cytoplasmic only Staining limited to cytoplasm Positive/negativePercent positiveIntensity
Membrane only Staining limited to cell membrane Positive/negativePercentage positive
Nuclear and cytoplasmic Staining in both nucleus and cytoplasm Positive/negativePercent positiveIntensity
Nuclear or cytoplasmic Variable location Cytoplasmic or nuclearPercent in a location
Membrano-cytoplasmic Staining in the cell membrane and the cytoplasm Positive/negativePercent positiveIntensity
Modified and used with permission by the Clinical and Laboratory Standards Institute, document I/LA28-A2.149
Table 7. Immunohistochemical Score Based on Different Percentagesof Cytoplasmic Staining.
Score Interpretation Description
0 0% No staining1 Borderline/background Weak staining in less than 50% of cells2 Positive Strong staining in less than 50% of
cells or weak staining in more than50% of cells
3 Strongly positive Strong staining in more than 50% ofcells
Modified and used with permission by the Clinical and Laboratory StandardsInstitute, document I/LA28-A2.149
Table 8. Scoring Approaches for Immunohistochemistry NuclearStaining.
0–4 Grading Scheme(% Positive Cells)
0–3 Grading Scheme(% Positive Cells)
0 (0) 0 (0)1 (1–25) 1 (1–10)2 (26–50) 2 (11–50)3 (51–75) 3 (51–100)4 (76–100)
Modified and used with permission by the Clinical and Laboratory StandardsInstitute, document I/LA28-A2.149
Ramos-Vara and Miller 71
granules, and other organelles or structures. An antigen may be
found in several locations, in several different organelles, in both
the nucleus and cytoplasm, and in the cytoplasmic membrane
and cytoplasm. Outside the cell, connective tissue, tissue
matrices, extracellular fluids, and other substances both normal
and abnormal can be immunoreactive. Antigens can exist in cer-
tain anatomical locations in normal cells but in other locations in
neoplastic or reactive cells. Genetic changes (eg, mutations) can
result in antigenic shift and redistribution; for instance, b-catenin
is typically a cell membrane protein but can be detected in the
nucleus in some cases.27,424 Antigens that exist at low levels
or have a short half-life in normal cells (eg, p53) may only be
detectable by IHC after mutation.
Patterns of Nonspecific Labeling. Nonspecific background
staining can mimic specific immunoreactivity. At the highest
antibody concentration, all cells (epithelial, mesenchymal, and
neural) and tissues are brown (with DAB as chromogen) (Fig.
20). With increasing dilution, nonspecific staining disappears.
Some cell types, especially mast cells and plasma cells, may
appear brown at all dilutions depending on the IHC technique,
but these cells will also be brown in the negative control slides,
which were not exposed to the primary antibody. The careful
use of appropriate controls should allow distinction between
background and specific labeling.
Immunocytochemistry
ICC is the detection of antigens in cytologic preparations by
means of an immunologic reaction visualized by a chemical
reaction.345 More biomarkers are immunoreactive in cytologic
preparations than in FFPE tissue sections.379 There are impor-
tant methodological differences between IHC and ICC in (1) the
type of substrate or sample, (2) storage of unstained samples,
(3) fixation, and (4) test validation and controls.86
Type of Cytologic Preparation. ICC can be performed on cytospins,
cell smears, cell blocks, cytoscrape cell blocks, cell cultures, and
liquid-based monolayer preparations.43,62,86,101,345,435 Cell
smears give reproducible results with nuclear markers but are
less suitable for cytoplasmic and membrane markers due to the
high background produced by cell damage during slide prepara-
tion.345 Cytospin preparations are less susceptible than smears to
cell damage.284 The use of pretreated slides to promote cell
adhesion reduces loss of cells during the ICC procedure.86
Liquid-based cytology using ThinPrep preparations enhances
retrieval of cells from small samples with preservation of cellu-
lar detail and, theoretically, a reduction of background due to
less blood, mucin, and proteinaceous material in the sample.86
Cell blocks are one of the best choices for ICC, particularly when
cells are numerous.239,285,286 Cell blocks are processed similarly
to surgical pathology specimens, so IHC methods can be used
without modifications.86 In addition, multiple sections can be
produced from a single block, so multiple markers can be
evaluated in the same cell population.86,286,394 However, cell
blocks can vary in cellular density and detail, depending on the
nature of the lesion, the quality of the fine-needle aspirate, and
postprocedural handling of the needle-rinse specimen.312 Impor-
tantly, because cell blocks are fixed in formaldehyde-containing
solutions, sections from cell blocks may need to undergo AR
procedures.45,339 A more detailed discussion of the pros and cons
of cytologic preparation methods is in Fowler and Lachar.107
ICC can be performed, even repetitively,252 on smears
previously stained with Romanowsky or Papanicolau and in
decolorized smears.1,14,240 However, cell loss, cell disruption
(affecting mostly cell membrane and cytoplasmic markers),
and antigenic degradation due to repeated passage through
graded alcohols can cause suboptimal results. In cases with
only 1 slide available and multiple markers to test, the sample
is divided into zones and a different antibody is applied to each
zone; alternatively, the sample can be divided using tissue
transfer techniques.329 Cell transfer techniques allow the eva-
luation of multiple markers when few slides are available.86
If cell transfer from previously stained cytologic smears is
anticipated, nonadhesive treated slides should be used.239
Cytologic Slide Storage. Storage of air-dried preparations for up to
2 weeks at 2�C to 8�C before ICC does not appear to reduce
their antigenicity.100 If longer storage is needed, slides should
be kept at –70�C.345,362 Some laboratories fix cytologic
samples with methanol and, if not used immediately, cover them
with 3% polyethylene glycol for storage or transportation.188
Fixation and Antigen Retrieval. Another difference between IHC
and ICC is the type of fixation. In contrast to IHC, in which
10% formalin is used routinely, there is no standard fixative for
cytologic specimens. In general, the type of fixation is deter-
mined by the cytological procedure and the antigens to be
tested.345 In ICC, samples are wet-fixed or air-dried and fixed
immediately before performing ICC with 100% acetone or
10% formalin.73,379 Air-dried preparations tend to lose fewer
cells than wet-fixed preparations, but inconsistent ICC results
have been reported with air-dried samples.86 For nuclear anti-
gens, fixation of air-dried specimens in buffered 4% to 10%formalin alone or followed by methanol-acetone produces
excellent results,345,361 although air-drying of smears before
fixation has been reported to hinder estrogen or progesterone
receptor detection.239 For membrane and cytoplasmic antigens,
the type of fixation is not as critical as for nuclear antigens;
various fixatives, including formalin followed by ethanol, a
1:1 mixture of methanol-absolute ethanol, or acetone at –
20�C, produce good results.345 However, acetone solubilizes
cell membranes, leading to diffusion of small peptides out of
cells and false-negative results (Fig. 6).383 Antigens such as
S100 protein, Hep Par 1, and gross cystic fluid protein 15 are
leached by alcohol fixatives, producing false-negative
results.62 One laboratory proposes these guidelines: samples
should be fixed immediately prior to the ICC procedure; for
lymphoid and melanoma markers, samples should be fixed for
5 to 10 minutes at RT in acetone; for epithelial markers, 5 min-
utes at RT in 95% ethanol or 1:1 mixture of methanol and 100%ethanol; and for nuclear antigens, 3.7% buffered formalin for
72 Veterinary Pathology 51(1)
15 minutes.100 Formal saline has been proposed as a ‘‘univer-
sal’’ ICC fixative.207 Others advocate air-drying slides if a
lymphoma is suspected and immediately fixing smears in
95% ethanol (without air-drying) in all other cases.239 A recent
comparison of FFPE cell blocks (CBs) with alcohol-fixed
centrifuged preparations (AFCPs) did not find significant dif-
ferences.166 Cytologic preparations fixed in acetone may be
more susceptible to cell loss than those fixed in formalin when
using automatic stainers because of weaker cytoplasmic attach-
ment to the glass slides.
AFCPs without AR performed similarly to cell blocks with
AR in many cases; however, for some tests, particularly to detect
nuclear antigens, AR has improved the reactivity of AFCPs.166
HIER using citrate buffer pH 6.0 is necessary for most nuclear
antigens on ethanol-fixed cytologic smears; for some cytoplas-
mic membrane and cytoplasmic antigens, it enhances the immu-
noreaction.80 Few cytoplasmic membrane antigens require
HIER with higher pH buffers.80 Some laboratories perform
HIER irrespective of the fixation type.435 Enzymatic AR is much
less commonly used than HIER in ICC.435 The mechanism of
action of HIER might depend on the fixative. When the amount
of cytologic specimen is limited, the same slide can be tested for
a second marker if the first test is negative.74 Adhesive slides
should be used to perform HIER or protease AR on smears; oth-
erwise, the sample is likely to detach.239
Immunocytochemical Method. Immunocytochemical procedures
are similar to those for IHC.286 Endogenous peroxidase is blocked
with 3% hydrogen peroxide; this step is unnecessary in acetone-
fixed samples. In blood-rich smears, the use of alkaline
phosphatase procedures rather than immunoperoxidase avoids
the background produced by endogenous peroxidase without the
need for strong quenching reagents.86 Antigen retrieval is often
necessary even without formalin fixation. Unfortunately, the
wide range of fixation procedures makes interlaboratory
standardization of AR difficult; each laboratory must optimize the
procedure for each antigen.188,286,341 Remarkably, in a multi-
institutional study, IHC quality was good irrespective of the
cytologic preparation or fixation procedure, except that cell block
preparations consistently had the highest scores.188
Controls and Standardization in Immunocytochemistry. Other
challenges in ICC are (1) use of adequate positive and negative
controls (ideally, controls should be treated in the same way as
test samples), (2) nonstandardized methodology and antibody
titers, (3) difficulty in evaluation of immunoreactivity in
inflamed or necrotic samples, (4) difficulty in distinguishing
normal from neoplastic cells, and (5) deleterious effect of
protease digestion in non–formalin-fixed material.107,188
Positive and negative controls are standardized in human
and veterinary IHC284,286 and should be included with each test
sample in ICC.62 Although the ideal tissue control is a compar-
ably fixed cytologic preparation,62 only 13% of publications
listed positive and negative controls processed identically as
the samples; 54% did not mention the use of controls or pro-
cessed controls separately.64 The College of American
Pathologists recognizes the impracticality of maintaining
separate positive control samples for every possible combina-
tion of fixation, processing, and specimen type (see comment
for questions ANP 22550 in the Anatomic Pathology checklist
at http://www.cap.org/apps/cap.portal). Cytologic control pre-
parations fixed in acetone lose antigenicity after several months
even if wrapped in aluminum foil and refrigerated; control
samples fixed in formalin retain their antigenicity indefi-
nitely.379 The production of cytologic controls from organs
(eg, lymph node, liver) is described elsewhere.379
Interpretation. As in IHC, the interpretation of ICC requires
consideration of the ‘‘antibody personality profile’’ and the
‘‘infidelity’’ (cross-reaction with antigens in other cell types)
of tumor-specific markers.425 First, a positive or negative
reaction must be defined.286 With the general lack of clear
guidelines, each laboratory should document and state in the
ICC report what is interpreted as a positive result. Alterna-
tively, as recommended in human IHC,124 a statement in the
ICC report indicating the intensity of the labeling and percent-
age of positive cells (of the type of interest) is more informative
than just a positive versus negative result. As for IHC, interpre-
tation of ICC tests should be done in conjunction with a
standard cytologic stain (eg, Wright-Giemsa, Papanicolaou)
and in concert with the clinicopathologic correlations.62
Causes of false-positive results in ICC include inadequate
fixation, insufficient blocking of endogenous peroxidase or
biotin activities, and high background labeling with PAbs, or
detection of immunoreactivity in the wrong cell type (eg, nor-
mal vs neoplastic).62,239,345 Two other causes of false-positive
results are spurious staining of cells that have phagocytized
other cells and reagent trapping in clusters or layers of cells.239
False-negative results are caused by improper fixation,
inadequate antibody titration, insufficient AR, or cell damage
during preparation.62,345
The Future of Immunohistochemistry
Since IHC was developed in the 1940s,65 major advances have
been made in the sensitivity and specificity of the method and
the availability of biomarkers for FFPE tissues. The main pur-
pose of IHC has been to characterize a neoplasm or identify an
infectious agent. However, the current trend is to apply IHC to
the detection of genetic abnormalities in neoplasms, detect
findings of prognostic importance, and aid in selection of
agents for cancer treatment.208 These topics are actively inves-
tigated in human medicine and not without controversy. Before
applying IHC to such investigations, the entire process must be
standardized, from the time a sample is taken from the patient
(preanalytical variables) through the analytical steps and
ending with the IHC results and diagnosis (postanalytical vari-
ables).|| Standardization of IHC procedures will be necessary to
compare results among laboratories and will be mandatory if
||References 10, 85, 208, 223, 308, 343, 369.
Ramos-Vara and Miller 73
computer-assisted quantification is intended, particularly if
those results influence the therapeutic approach. Although the
barriers to progress are daunting, the in situ detection of protein
expression by IHC will be a vital part of pathology for years to
come, even if the generation, interpretation, and application of
test results in the context of disease change drastically.
Acknowledgements
We thank Marilyn Beissenherz, VMDL–University of Missouri, and Dee
DuSold, ADDL–Purdue University, for their dedication to preparing
IHC slides and troubleshooting. We also thank Dr Chang Kim, Compara-
tive Pathobiology, Purdue University, West Lafayette, Indiana, and Dr
van der Loos, Academic Medical Center, Amsterdam, the Netherlands,
for reviewing portions of this manuscript. Finally, we thank the veterinar-
ians who submitted cases for IHC to the laboratories in which the authors
have worked and learned the red, brown, and blue technique.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, author-
ship, and/or publication of this article.
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