When Tissue Antigens and Antibodies Get Along

Effects of Delayed Fixation

Prompt transfer of postmortem specimens 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 intestine or pancreas, autolyze rapidly; proteolysis can lead to increased background staining (Fig. 2). ¹⁴⁹ Diffusion of 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 fixative; 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 (noncoagulating) 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 versus background (signal-to-noise ratio), and even the detection 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 contain 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 carbohydrates. ^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 simultaneously, they do so at different rates, with penetration being about 12 times faster than bonding and the latter 4 times faster 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) produces the characteristic cross-linking formaldehyde fixation. ^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 sensitivity 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 eventually 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.

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 proteins. ^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 fixation but also by postfixation procedures. ¹⁰⁸

The deleterious effects of formaldehyde on immunoreactivity in cells and tissues can be partially reversed, ⁹² whereas glutaraldehyde fixation is considered irreversible. ⁹¹ Prolonged 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, therefore, facilitates antigen detection should the tissues be needed subsequently for IHC. Overfixation can also be partially corrected 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 (-NH₃ ⁺) 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 (-NH₃ ⁺) 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) produces more cross-links than nonbuffered formalin and therefore more deleterious effects for IHC. ²⁸⁴ If the fixative is acidic formalin, cross-linking is reduced, so the antigen retrieval 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 preserving antigenicity. ⁴¹²

After an initial drop in immunoreactivity with fixation, there is a plateau of variable duration before antigens become unrecognizable 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 procedure. ^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 localization ¹⁴² —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 overfixation. ^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 coagulation 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 biomarkers. ^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 subsequent 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 immunoreactivity. 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 recommended 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 Fixative 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 α-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, trichloroacetic 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 preservation ^142,153,401 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 cytoplasmic 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 postfixation stabilization reagent (alcohol mixture), may not need antigen 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 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 different 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 microwaves and, because of the release of formalin vapors, must be performed in a fume hood.

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 cleavage 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 commercially 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 number 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 introduced by Shi et al. ³³⁷ based on a concept developed by Fraenkel-Conrat et al ^110–112 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 hydrolysis 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, antigens 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 substances (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 antigens, 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 longer 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 morphology 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 β. ¹⁷⁹

The degree of fixation can dramatically modify the response of antigens to AR. Unfixed proteins are denatured at temperatures 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 disputed, 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).

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Figure 6.

Effects of fixation on small peptides and cell membrane antigen stability. Formalin fixation stabilizes small peptides and cell membrane antigens, preventing leakage or diffusion, even after sectioning. With acetone fixation, small peptides leak through the permeabilized cell membrane. Formalin fixation stabilizes membrane antigens in cryostat sections, but small peptides leak away. Used with permission and modified from Floyd and van der 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 (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 could preclude 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 HIER alone (upper inset). Immunoperoxidase-DAB, hematoxylin counterstain.

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Figure 10.

Loss of immunoreactivity after formalin fixation with recovery by antigen retrieval (AR). (a) Putative structural changes in native proteins. (b) Corresponding changes to epitopes in the in vitro peptide assay. Used with permission from Sompuran et al. ³⁴⁷

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 composition. ³⁹ The possibility of unexpected immunoreactivity 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 compared ³³¹ 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 protected amines and liberating formaldehyde quickly to avoid reforming adducts; then citraconic acid liberates amines via hydrolysis. ²⁵³

Detergents, although not strictly speaking an AR component, 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 usually 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 procedure 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 deparaffination, 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 surfactants 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 affecting the IHC reaction. ¹¹⁴

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 (homopolymeric) 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) isoforms, 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 preparations. 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 peptide 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 modifications of the native antigen are absent in synthetic peptides. ^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 However, protein purification to homogeneity from cells or tissues can be technically difficult, and contaminating proteins may be more antigenic than the protein of interest, producing a disproportionate 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 differ markedly in amino acid sequence. Unfortunately, the source and details of antigen purification or the isoforms of the targeted protein recognized by an antibody preparation are seldom reported for commercial primary antibodies. ²³⁷ Other factors affecting immunogenicity include size, conformation, and electrical 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, κ and λ. In any immunoglobulin molecule, 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 C_L (Constant: Light chain), and the N-terminal half, which has abundant sequence variability and is called the V_L (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 techniques ²⁸⁴ and is largely responsible for nonspecific background reactivity due to nonimmune adherence of antibodies (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 terminus. 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 binding 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 component 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 conformational folding or binding. ³⁹ Although most epitopes involved in immune responses are believed to be conformational, 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 electrostatic) 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 phenylalanine, 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 electrostatic attractions are maximal at the shortest distances. Therefore, 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 C=O, NH and C=O, and NH and OH groups with binding 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 configuration 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.

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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 buffer
Electrostatic	Neutral pH of buffer	Extreme pH of buffer
	Low ionic strength of buffers	High ionic strength buffer
	Low incubation temperature	High incubation temperature

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 antigenic determinant (epitope). ²¹³ The affinity constant (K_a) represents the amount of antibody-antigen complex formed at equilibrium.

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K a = [ A b ~ A g ] [ A g ] ⋅ [ A b ]

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 K_a of 10¹² L/mol has 1000-fold greater affinity than one with a K_a of 10⁹ L/mol). Affinity can be determined precisely only for monoclonal antibodies (MAbs); ²¹³ for polyclonal antibodies (PAbs), composed of antibodies with different affinities, affinity can only be estimated. Another method currently used by antibody manufacturers to calculate the affinity of antibodies is determining the equilibrium dissociation constant (K_d). Most antibodies have K_d values in low micromolar (10^–6) to nanomolar (10^–7 to 10^–9); very high affinity antibodies have picomolar (10^–12) values. In other words, a high K_a value (eg, 10¹²) will correspond with a very low K_d value (eg, 10^–12). New high-throughput technologies such as microarray-based kinetic constant assays ^201,202 allow the K_d 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 reaction 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

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 mammalian 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 purified, 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 epitopes. 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 antigen detection by recognition of multiple epitopes, can be a disadvantage: 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 Köhler 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 produce immortal hybrid cells that produce Igs specific for a single epitope (MAbs). The immortalized hybrid cells form hybridomas 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 eliminate 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

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Table 4.

Comparison of Polyclonal and Monoclonal Antibodies for Immunohistochemistry.

	Polyclonal Ab	Monoclonal Ab
Origin	Multiple species	Mouse, rabbit
Total 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 lines
Epitope recognition	Multiple, including multiple isoforms of the same protein	One single epitope
Specificity	Low to high	High
Affinity	Variable (both high and low)	Homogeneous
Avidity	High	Lower than polyclonal antibodies
Fixation tolerance	High	Variable to low
Fixation effects	Multiple	Single
Molecular 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

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Table 5.

Standard Immunohistochemical Protocol Using a 2-Step Polymer-Based Detection System.^a

1. Deparaffinize slides and bring them to deionized water

2. Antigen retrieval (eg, enzyme, HIER) procedure, if needed

3. Rinse in deionized water

4. Block endogenous enzyme activities (eg, peroxidase)

5. Rinse sections and bring them to buffer

6. Nonspecific staining blocking

7. Remove excess of blocking solution (do not rinse)

8. Incubate section with primary antiserumb

9. Rinse slides with buffer

10. Incubate with secondary reagent (immunoglobulin anti–primary antibody species)

11. Rinse slides with buffer

12. Incubate slides with tertiary reagent (enzyme-immunoglobulin-polymer complex)

13. Rinse slides with buffer

14. Detection of the immune reaction with developing reagents (eg, DAB and H2O2 for peroxidase)

15. Rinse sections with deionized water

16. Counterstain with hematoxylinc

17. Dehydrate and coverslipd

HIER, heat-induced antigen retrieval.

^aThe best incubation times for the primary antibody and other reagents should be based on information provided by manufacturers of various reagents 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 and error. The incubation chamber should have enough moisture to avoid dessication of the slides.

^cSelect carefully the type of counterstain based on the chromogen used and location of the antigen.

^dUse water-based coverslipping media when the precipitate of the enzymatic reaction is sensitive to organic solvents.

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 glycosylation), 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 targeting 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 Staphylococcus aureus (A) and group G (G) streptococcal strains ⁴⁶ 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 specific 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 immunoglobulins) 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 available 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 section. ^116,152,227 Secondary antibodies to mouse IgGs may also react with Igs of other species producing the same type of nonspecific background in plasma cells, serum, or any tissue containing 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?

Specificity 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. Polyclonal 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 culture or polymerase chain reaction (PCR). Nonspecific binding 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 Kalyuzhny ¹⁸¹ wrote, “Having a good antibody for immunohistochemistry 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.”

How to Find the Antibody That Matches Your Needs

Antibodies are selected based on consultation with other scientists, publications, and information from manufacturers. ⁴⁷ Useful Internet resources include the Human Protein Atlas (http://www.proteinatlas.org/), which contains information on protein and subcellular 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 reactivity of the antibodies in tissue sections. Other sites with information on commercial antibodies are http://www.antibodypedia.com, http://www.biocompare.com, http://antibodyresource.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 Immunohistochemistry Database (http://ncifrederick.cancer.gov/rtp/lasp/phl/immuno/) lists many biomarkers with specific protocols for IHC in mouse tissues.

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 sensitivity for the detection of most antigens in routinely processed tissues (Fig. 11).

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Figure 11.

Direct and indirect immunohistochemistry (IHC) methods. Figure 12. Avidin-biotin complex (ABC) method. Figure 13. Labeled streptavidin-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: LSAB+ (a, b), a universal detection system labeling rabbit, mouse, and goat primary antibodies; EnVision+ (ENV+) (c, d), a polymer detection system labeling only mouse primary antibodies. Upper panel: Carcinoma, skin; dog. Minimum background with LSAB+ (a) and none with EnVision+ (c). Lower panel: Mammary gland; goat. Extensive background with LSAB+ (a) due to binding to endogenous goat immunoglobulins. No background when using EnVision+ (c). (b, d) Negative reagent controls. Immunoperoxidase-DAB for cytokeratins using a mouse monoclonal antibody, hematoxylin counterstain. Figure 18. Double immunolabeling using 2 mouse monoclonal antibodies with same class isotype immunoglobulin. Figures 11 to 16 and 18 are modified from Ramos-Vara, ²⁸⁴ with permission from Veterinary Pathology.

Indirect Methods

The need for more sensitive antigen detection prompted Coons et al ⁶⁷ 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 sensitivity is higher than with a direct method because (1) the unlabeled primary antibody retains full avidity with stronger Ag 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 antigen 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

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 (peroxidase-antiperoxidase) of 2 rabbit IgG molecules with 3 peroxidase 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 methods, 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 secondary 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 avidin. ^{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 systems (eg, EnVision, PowerVision, ImmPRESS, MACH 4^M), which are the method of choice in many laboratories. The sensitivity 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 sensitivity, lower background, and reduced nonspecific binding. ¹⁴⁹

Catalyzed Signal Amplification

This method is based on the ability 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 tyramide at the location of the antigen-antibody reaction. Free radicals formed during the HRP-tyramide reaction bind covalently to electron-rich amino acids (eg, tyrosine) of nearby proteins. ⁴⁸ The reactive intermediates are so short-lived that biotinylated tyramide is deposited only at or near its site of production. 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 treatment to quench endogenous peroxidase or EABA is usually necessary. Modifications using fluoresceinated tyramide result in marked reduction or ablation of background from endogenous biotin. ^{139,140,259,389}

Immuno-Rolling Circle Amplification

Rolling circle amplification (RCA) increases the signal of the immunologic reaction without 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 polymerase, 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 complementary 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 isothermal 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 polyvalent (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 incompatibilities 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 methods, 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 different components of the reaction. This requires careful planning and the use of multiple controls. Double immunolabeling methods can be simultaneous (parallel) or sequential. ³⁸² Simultaneous 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 solutions. ¹⁴⁹ As a rule, if the primary antibodies are from the same species, sequential dual labeling is necessary. The risk with 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 antigens 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 antibody 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 antigens 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, presence 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 peroxidase, 3,3′-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, permanent media) are the usual chromogens. ²⁸⁴ Alkaline phosphatase is recommended for immunocytochemistry. ³¹ Van der Loos ³⁸⁵ 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.

“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 without “online” HIER. Several platforms are designed for continuous 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 testing. Other platforms, especially those with ISH capability, can use higher incubation temperature (eg, 37°C) to increase reaction speed. Reagent volume may determine the cost of a test; some platforms require about 100 μl 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 customize 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 quality of immunostains in many laboratories, the machines are not perfect, and it is naïve to assume that obtaining an automated instrument will guarantee quality and reproducibility.” ²³⁹

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 antibody 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 immunoreactivity 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 morphology and location of the labeled organisms supports IHC results. For cell markers, reactivity should be detected in the appropriate cell or tissue compartment (Fig. 19). More information on primary antibody evaluation is in the section on test validation.

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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 be expressed in a paranuclear (Golgi) location. Inset: Detail of the reaction. (d) Plasmacytoma, skin; dog. The transcription factor MUM1 is expressed in plasma cell nuclei. Inset: Detail of reaction. Immunoperoxidase-DAB, hematoxylin counterstain. Figure 20. Optimal titration of the primary antibody (Ab). Lymph node; dog. Prox-1 is a transcription factor of lymphatic endothelial cells. (a) Excess Ab (1/100 dilution) results in muddy nuclear labeling in both endothelial cells and lymphocytes with background in plasma (v). (b) Specific nuclear labeling of Prox-1 in lymphatic endothelium at 1/400 Ab titer. (c) Labeling of lymphatic endothelial cells (arrowheads) with lack of background in lymphocytes or plasma (v) at 1/800 titer. Note hemosiderin in macrophages at bottom (asterisk). Immunoperoxidase-DAB, hematoxylin counterstain. Figure 21. Antibody standardization for immunohistochemistry (IHC). Three sets of sections (5 slides per set) are used. Two-fold dilutions (1/25 through 1/200) were made (first 4 slides on each row; the last slide is the negative reagent control). Sections in 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 antibodies (MAbs) to vimentin. (a) Mouse MAb reacted appropriately with mesenchymal cells such as lymphocytes (arrowheads), smooth muscle cells of vessels (asterisk), and interstitial cells (solid circle) and did not label epithelial cells. (b) Rabbit MAb had no reactivity. Immunoperoxidase-DAB, hematoxylin counterstain.

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 demonstrate 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 posttranslational modifications or low antibody specificity. ^40,200,321 Nevertheless, WB immunoreactivity does not necessarily predict immunoreactivity in FFPE tissues; WB requires antigen denaturation (therefore, modification of its secondary and tertiary structure) as opposed to the in situ antigen modification in formalin-fixed tissues, with cross-linking rather than denaturation. ^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 procedures 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 K_d 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 capabilities 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 immunoreactivity 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 appropriate 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 definition, 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 tissue 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 biologically 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 different company produced the expected results (Figs. 23, 24). This discrepancy suggests that nonactive ingredients of the monoclonal preparation could have interfered with the immunologic reaction.

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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 the submucosa (s) or muscularis (m). (b) Detail of the mucosa. (c) Detail of the submucosa and muscularis. Immunoperoxidase-DAB, hematoxylin counterstain. 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 proprial leukocytes (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 marker PNL2 labels neoplastic cells of equine melanoma. (b) Melanocyte marker Melan A is not expressed in equine melanoma. Insets: Immunohistochemistry 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 have nonspecific 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 unexposed to HIER solution, therefore unable to react with the primary Ab. (b, c) Reactivity was minimal in the portion of the section not exposed to HIER solution (solid circle). Immunoperoxidase-DAB for equine herpesvirus 1, hematoxylin counterstain.

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

Effects of Fixation, Postfixation Treatments, and Equipment on Immunoreactivity

These laboratory variables affect assay development and validation. ⁴¹⁹ Different fixatives have different effects on immunoreactivity. For each fixative, full standardization 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 reproducible 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 conditions 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 intralaboratory 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 Immunoreactivity

The pattern of immunoreactivity, particularly with neoplasms, 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 however, cytoplasmic TTF1 expression has been reported in human liver neoplasms and confirmed in mitochondria by other methods. ^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}

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 routinely 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 differently. When possible, the positive tissue control should be on the same slide as the test tissue to minimize technical variation.

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 background (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 multitissue blocks for antibody validation studies or as tissue controls, 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 control 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 erythrocytes. 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 variety 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 antibody is replaced by 1 of the following: (1) antibody diluent, (2) same-species nonimmune immunoglobulin at the same concentration 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 commercially 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 forthcoming. 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, separate controls should still be run for each different protocol (eg, different AR or detection system). ³⁶⁹

Daily Quality Assurance/Quality Control

Each laboratory should establish standard operating procedures (SOPs) for histology. These include schedules for cleaning, maintenance, and monitoring 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’ recommended 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 interlaboratory 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 animal 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 Fletcher ¹⁰⁴ 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

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 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 certain 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, β-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.

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 preparation. 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 cellular 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 Importantly, 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 evaluation 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 determined 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 antigens, 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 minutes 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 “universal” 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 differences. 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 attachment 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 cytoplasmic membrane and cytoplasmic antigens, it enhances the immunoreaction. 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; otherwise, 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 IHC ^284,286 and should be included with each test sample in ICC. ⁶² Although the ideal tissue control is a comparably 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 processed controls separately. ⁶⁴ The College of American Pathologists recognizes the impracticality of maintaining separate positive control samples for every possible combination 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 preparations fixed in acetone lose antigenicity after several months even if wrapped in aluminum foil and refrigerated; control samples fixed in formalin retain their antigenicity indefinitely. 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. Alternatively, as recommended in human IHC, ¹²⁴ a statement in the ICC report indicating the intensity of the labeling and percentage of positive cells (of the type of interest) is more informative than just a positive versus negative result. As for IHC, interpretation 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, normal 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