When Tissue Antigens and Antibodies Get Along

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published online 15 October 2013Vet PatholJ. A. Ramos-Vara and M. A. Miller

The Red, Brown, and Blue Technique−−ImmunohistochemistryWhen Tissue Antigens and Antibodies Get Along: Revisiting the Technical Aspects of

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Article

When Tissue Antigens and Antibodies GetAlong: Revisiting the Technical Aspects ofImmunohistochemistry—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 tar-

get 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 par-

ticular 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 Pathology00(0) 1-46ª The Author(s) 2013Reprints and permission:sagepub.com/journalsPermissions.navDOI: 10.1177/0300985813505879vet.sagepub.com

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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.

2 Veterinary Pathology 00(0)




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.

Ramos-Vara and Miller 3




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.





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





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.





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.





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.





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





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





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.





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





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





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





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





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





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.





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.





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.





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





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.





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





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)





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.





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





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.





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





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





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





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 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

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





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





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.





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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