Immunohistochemical and immunofluorescent techniques

Immunohistochemistry (IHC)

A technique for identifying cellular or tissue constituents, antigens, by means of antigen-antibody interactions. The site of antibody binding is identified either by direct labeling of the antibody or by use of a secondary labeling method.

Antigen

An antigen is a molecule which induces the formation of an antibody and bears one or more antibody-binding sites. These are highly specific topographical regions composed of a small number of amino acids or monosaccharide units known as antigenic determinant groups or epitopes.

Antibody

Antibodies belong to the class of serum proteins known as immunoglobulins. The terms antibody and immunoglobulin are often used interchangeably. They are found in blood and tissue fluids, as well as many secretions. The basic unit of each antibody is a monomer; antibodies can be monomeric, dimeric, trimeric, tetrameric, or pentameric in format. The monomer is composed of two heavy and two light chains. If it is cleaved with enzymes such as papain and pepsin, two fragment binding antigens (Fab) fragments and a crystallizable fragment (Fc) are produced. They are formed in the humoral immune system by plasma cells, the end cell of B-lymphocyte transformation, after recognition of a foreign antigen. There are five types of antibody found in the blood of higher vertebrates: IgA, IgD, IgE, IgG, and IgM.

IgG is the most common and frequently used antibody for IHC. The IgG molecule is composed of two pairs of light and heavy polypeptide chains linked by disulfide bonds to form a Y-shaped structure. The terminal regions of each arm vary in amino acid sequence and are known as ‘variable domains’. This variability in amino acid content provides specificity for a particular epitope and enables the antibody to bind specifically to the antigen against which it was raised.

Antibody-antigen binding

The amino acid side-chains of the variable domain of an antibody form a cavity-like site which is geometrically and chemically complementary to an antigen epitope molecule, as described by . The analogy of a lock (antibody) and key (antigen) has been used, and the precise fit required explains the high degree of antibody-antigen specificity seen. The associated antibody and antigen are held together by a combination of hydrogen bonds, electrostatic interactions, and van der Waals’ forces.

Affinity

Affinity is the three-dimensional fit of the antibody to its specific antigen, and is a measure of the binding strength between the antigenic epitope and its specific antibody-combining site.

Avidity

Avidity is a related property referring to the heterogeneity of the antiserum which will contain various antibodies reacting with different epitopes of the antigen molecule. A specific but multivalent antibody is less likely to be removed by the washing process than a monovalent antibody. Avidity therefore is the functional combining strength of an antibody with its antigen.

Antibody specificity

This is the characteristics of an antibody to bind selectively to a single epitope on an antigen.

Sensitivity

This is the relative amount of antigen which an IHC technique is able to detect. A technique with high sensitivity is able to detect smaller amounts of antigen than a technique with low sensitivity. If used to detect the same amount of antigen, the technique with high sensitivity would produce a larger signal than a method with low sensitivity.

Polyclonal antibodies

Polyclonal antibodies are produced by immunizing an animal with a purified specific molecule, an immunogen, bearing the antigen of interest. The animal will mount a humoral response to the immunogen and the antibodies produced can be harvested by bleeding the animal to obtain immunoglobulin-rich serum. It is understood that numerous clones of plasma cells will be activated to produce the polyclonal antibodies.

Each clone will produce an antibody with a slightly different specificity to the variety of epitopes present on the immunogen. A polyclonal antiserum is therefore a mixture of antibodies to different epitopes on the immunogen. Some of these antibodies may cross-react with other molecules and will need to be removed by absorption with the appropriate antigen. The antiserum will probably contain antibodies to impurities in the immunogen. Antibodies raised against the contaminating immunogens are often of low titer and/or affinity, and can be diluted out to zero activity for immunolabeling. There is a high possibility that a wide spectrum of naturally occurring antibodies will be present in the host animal as a response to previous antigen challenges. Serum removed from the animal, before injection of the immunogen, is therefore important as a negative or pre-immune control. For precise details of polyclonal antibody production see .

Monoclonal antibodies

The development of the hybridoma technique by to produce monoclonal antibodies has revolutionized IHC by increasing enormously the range, quality and quantity of specific antisera. Detailed descriptions of the technique have been given by and .

The method combines the ability of a plasma cell (transformed B lymphocyte) to produce a specific antibody with the in vitro immortality of a neoplastic myeloma cell line; a hybrid with both properties can be produced. With the technique of cloning, this cell can be grown and multiplied in cell culture theoretically to unlimited numbers. By careful screening, hybrids producing the antibodies of interest, without cross-reactivity to other molecules, can be chosen for cloning. The original antigen does not need to be pure as hybrids reacting to unwanted antigens or epitopes can be eliminated during screening. The result is a constant, reliable supply of one pure monoclonal antibody with known specificity.

This approach to the production of monoclonals has dramatically increased the number of antibodies available for IHC and has allowed for further evolution with the ability to identify more antigens in paraffin wax sections. Detailed comparisons of the values and limitations of polyclonal and monoclonal antibodies have been given by and .

For immunofluorescent techniques a preparation of highly purified or recombinant antigen is an absolute requirement for the production of monospecific antiserum having a high affinity and avidity. Antibody specificity can be determined by reacting it against the purified antigen used to immunize the animal. It can also be tested against the unpurified source such as whole human serum, e.g. if the antibody is against a specific human protein. This checking for monospecificity can be achieved by gel diffusion, immunoelectrophoresis or passive hemagglutination, and should result in the production of one precipitin line in both the purified antigen and the unpurified source. Antibody concentrations of relatively high titer are required for conjugation with fluorochromes.

Enzyme labels

Enzymes are the most widely used labels in immunohistochemistry, and incubation with a chromogen using a standard histochemical method produces a stable, colored reaction end product suitable for the light microscope. In addition, the variety of enzymes and chromogens available allow the user a choice of color for the reaction end product.

Horseradish peroxidase (HRP) is the most widely used enzyme, and in combination with the most favored chromogen, 3,3α-diaminobenzidine tetrahydrochloride (DAB), it yields a crisp, insoluble, stable, dark brown reaction end product ( ). DAB is classed as a hazardous chemical and has been reported to be a potential carcinogen ( ).

Horseradish peroxidase is commonly used as an antibody label for several reasons:

• Its small size does not hinder the binding of antibodies to adjacent sites.

• The enzyme is easily obtainable in a highly purified form and therefore the chance of contamination is minimized.

• It is a stable enzyme and remains unchanged during manufacture, storage and application.

• Any endogenous activity is easily quenched.

Other chromogens are available, including: 3-amino-9-ethylcarbazole ( ), which gives a red final reaction product; 4-chloro-1-naphthol ( ), a blue final reaction product; Hanker-Yates reagent ( ), a dark blue product and α-naphthol pyronin ( ), a red-purple final reaction product. Many of these contain hazardous reagents and have now largely been superseded by commercial chromogens available in kit form.

Vector Laboratories, for example produce a wide range of different colored chromogens suitable as alternatives to DAB which can be used for multi-labeling techniques. These include Vector Red, Vector Blue, Vector VIP (purple) and BCIP/NBT(blue/violet).

It should be noted that some of these chromogens produce reaction products which are soluble in alcohol and xylene, and therefore the sections require aqueous mounting. Commercial products are now available which have improved preservation qualities and resolution compared with the traditional aqueous mountants. After drying in a hot oven, these mountants give a hard permanent covering of the section. Other commercially available permanent mounting media which are non-aqueous and both toluene and xylene free are also available (Vector Laboratories VectaMountTM), and they provide a permanent preparation for use with enzyme substrates such as Vector Red, Vector VIP and BCIP/NBT.

Endogenous peroxidase activity is present in a number of sites, particularly neutrophil polymorphs and other myeloid cells. Blocking procedures may be required, the hydrogen peroxide-methanol method ( ) being the most popular. Care should be taken with certain antigens, notably CD4, where too long an incubation in the blocking solution or too high a concentration of hydrogen peroxide can significantly diminish staining on formalin fixed, paraffin wax embedded tissue. Performing the peroxidase block after the binding of the primary antibody to the tissue antigen is to be recommended for antibodies such as CD4.

Calf intestinal alkaline phosphatase is the most widely used alternative enzyme tracer to horseradish peroxidase, particularly since the development of the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method by Fast red TR used with naphthol AS-MX phosphate sodium salt gives a bright red reaction end product which is soluble in alcohol. New fuchsin has been reported as giving a permanent insoluble red product ( ) when mounted in resinous mountant, but it is the experience of many workers in the field that the resistance of the reaction product to resinous mounting is inconsistent.

Endogenous alkaline phosphatase activity is usually blocked by the addition of levamisole to the substrate solution. Levamisole selectively inhibits certain types of alkaline phosphatase, but not intestinal or placental, when used at a concentration of 1 mM. Twenty percent glacial acetic acid is a better blocker of endogenous alkaline phosphatase activity as it inhibits all types of alkaline phosphatase.

Other labels such as glucose oxidase, bacterial-derived β- d -galactosidase , colloidal gold and silver metals have been used in the past, but failed to find a routine use in the diagnostic laboratory. Colloidal gold has a much wider use in the electron microscope field.

Fluorescent labels

Fluorochromes are fluorescent labels which when conjugated to the antibody absorb ultraviolet or visible light of a particular wavelength to reach an unstable excited state as its electrons gain energy. The fluorochrome subsequently emits light of a different, usually longer, wavelength to that of the excitation light as the electrons return to their ground state.

Serum proteins have differing capacities to combine or conjugate with fluorochromes. Immunoglobulins, in particular, have less affinity for fluorescein isothiocyanate (FITC) than other more ‘negatively charged’ proteins such as albumin and β-proteins. These latter molecules when conjugated can also combine with tissue components via electrostatic forces and give high levels of non-specific staining. Purified immunoglobulin free from other serum proteins is therefore a pre-requisite for conjugation. Immunoglobulin molecules are composed of heavy and light chains. The heavy chains identify the isotype of the antibody and the light chains are common to all immunoglobulin types. Consequently, an antibody raised to a particular immunoglobulin type may also cross-react with all other immunoglobulin types due to the presence of antibodies to the light chains. These contaminating light chain antibodies should be removed by absorption against free light chains and leave only antibody reacting against the heavy chain component, i.e. γ, α or μ chain specific.

Conjugates may be prepared from:

• 1.

  Immunoglobulin rich fractions of serum prepared by salting-out procedures.

• 2.

  Chromatographically prepared fractions, usually on diethyl aminoethane (DEAE) ion exchange columns, consisting mainly of IgG.

• 3.

  Pure IgG fractions obtained by immunoabsorption on affinity chromatography columns.

• 4.

  F(Ab)2 fractions of IgG obtained by proteolytic cleavage of purified IgG which has been obtained as in 3 above.

The four methods are progressive in terms of the purity of their antibody preparation, and each can be used with success in different situations. For most routine applications, reagents prepared using purification methods 1 or 2 are adequate, particularly when they can be used at high dilution. Reagents prepared with method 2 are useful when background staining is a problem. Conjugates made from F(Ab)2 fragments are used when the binding of the conjugated antibody to Fc receptors is to be avoided. They may also be useful in double staining techniques where cross-reactions between antibodies produced in different species are a problem.

The absorption and emission characteristics of several commonly used fluorochromes are summarized in Table 19.1 ( ). FITC is the most widely used fluorochrome in immunofluorescent microscopy. It has a wide absorption spectrum which covers the ultraviolet to blue light range and has a characteristic apple-green emission. An advantage of FITC is that the apple-green fluorescence is rarely seen as autofluorescence in mammalian tissues. Rhodamine absorbs maximally in green light and has an orange-red emission light; it can be used in a two color technique where two different antigens can be identified on the same section by antibodies conjugated with FITC or rhodamine.

The conjugation of a fluorochrome with an antibody can be a complex reaction and is dependent on the type of fluorochrome. FITC and rhodamine can be linked covalently to free terminal amino and carboxyl groups, free amino groups on lysine side chains and free carboxyl groups in aspartic and glutamic acid residues. The reactions occur at pH 9.5 and the degree of conjugation is both time and temperature dependent. The ideal fluorochrome to antibody ratio is between 2 and 4:1.

Over-conjugation of the antibody will give high background staining as the molecules have a net negative charge and will bind to tissue non-specifically. This can also result in poor reactivity of the antibody due to interference with antigen binding sites. Under-conjugation gives a preparation which will produce unsatisfactory low-level fluorescence.

Free chromophore in the conjugate preparation must be removed to prevent non-specific staining. The free chromophore can be removed by dialysis against 0.15 M sodium chloride at 4°C. The presence or absence of fluorescence in the dialysate can be seen under UV examination. An alternative is to use gel filtration column chromatography using Sephadex G50. A method for testing commercial conjugate preparations for free dye is described by .

It is good laboratory practice to evaluate the sensitivity and specificity of all antisera and conjugates used in immunofluorescence. In assessing the sensitivity of the reagents, a checkerboard test will indicate the optimal working dilution for a particular antibody or conjugate. For indirect immunofluorescence, serial dilutions of the antiserum or conjugate are tested against serial dilutions of the unlabeled primary antibody and the tissue sections assessed for the least amount of background fluorescence which still allows identification of the target antigen. The conjugate working dilution in direct immunofluorescence can be determined by examining serial dilutions of the conjugate on a tissue section containing known protein deposits e.g. renal sections from an IgA nephropathy or skin sections from known pemphigus or pemphigoid patients.

Specificity checking of a working strength conjugate on a tissue section with known deposits shows whether or not it will cross-react with other antigens. Anti-IgG γ-chain-specific conjugate for example, should not react with other classes of immunoglobulin such as IgA, IgM or kappa/lambda (κ/λ) light chains.

Some anti-animal immunoglobulin specific conjugates used in indirect immunofluorescence may cross-react with human immunoglobulins. In such cases it is essential that the cross-reacting antibodies are removed by absorption with human immunoglobulin. Cross-reactivity can be assessed by incubating the conjugated antibody directly on a tissue section containing human immunoglobulin and examining the slide for fluorescence.

Radiolabels

The use of radioisotopes as tracers requires autoradiographic facilities, and developed from the need for quantitation in IHC. Techniques involving the use of radioisotopes as tracers have been discussed by but are not used in the routine diagnostic laboratory.

Polymer chain two-step indirect technique

This technology uses an unconjugated primary antibody, followed by a secondary antibody conjugated to an enzyme (horseradish peroxidase) labeled polymer (dextran) chain ( Fig. 19.6 ). This dextran chain has up to 70 enzyme molecules and 10 antibody molecules attached. Conjugation of both anti-mouse and anti-rabbit secondary antibodies enables the same reagent to be used for both monoclonal (rabbit and mouse) and polyclonal (rabbit) primary antibodies. The method is biotin free and therefore does not react with endogenous biotin. In addition to being quick, reliable and easily reproducible, the technique offers great sensitivity. The technique is also useful for multi-color staining on single slide preparations. This technique is now the most commonly used method in routine diagnostic use. There is a wide choice of commercially available polymer kits, e.g. EnVision™ ⁺ and FLEX ⁺ from Dako, Novolink and Bond Polymer Refine from Leica, Immpress™ from Vector Laboratories, Excel + from Menarini and ultraVIEW from Ventana.

Unlabeled antibody-enzyme complex techniques (PAP and APAAP) and Immunogold silver staining technique (IGSS)

These methods are rarely found now in the diagnostic setting but descriptions of these techniques can be found in previous editions of this publication.

Proteolytic enzyme digestion

Pretreating formalin-fixed routinely processed paraffin wax sections with proteolytic enzymes to unmask certain antigenic determinants was described by and . The most popular of these employed today are trypsin and protease, but others such as chymotrypsin, pronase, proteinase K and pepsin may also be used. The theory behind the unmasking properties of these proteolytic enzymes is not fully understood. It is generally accepted however, that the digestion breaks down formalin cross-linking and hence the antigenic sites for a number of antibodies are exposed.

Proteolytic digestion can be detrimental to the demonstration of some antigens, occasionally producing false positive or false negative results. Digestion times need to be tailored to individual antibodies and to the fixation time. Under-digestion results in too little staining because the antigens are not fully exposed; over-digestion can produce false positive staining, high background levels and tissue damage. There can be a fine balance between under-digestion and over-digestion when using proteolytic enzymes. Duration of enzyme digestion, enzyme concentration, use of a coenzyme such as calcium chloride with trypsin, temperature and pH must be optimized to produce consistent high-quality IHC staining. Different batches of enzyme may vary in quality and each new batch of enzyme should be assessed prior to routine use. Enzymes produced specifically for IHC use are now widely available from commercial sources. These have been produced for use with automated immunostaining machines, are easy to use and give good consistent results.

The use of heat-induced epitope retrieval techniques has largely replaced proteolytic digestion. To demonstrate immunoglobulins and complement in formalin-fixed paraffin wax embedded renal biopsies and for a number of other individual antigens however, proteolytic digestion is still favored by many.

Microwave antigen retrieval

first established the use of microwave heating for antigen retrieval; the use of heavy metal salts posed a significant risk to the health and safety of the users. used microwave antigen retrieval with a non-toxic citrate buffer at pH 6.0 and demonstrated the Ki67 antigen which previously had been thought to be lost during formalin fixation and paraffin wax processing. The results were equivalent to those seen in frozen sections. established microwave oven heating as an alternative to proteolytic enzyme digestion. The method improved the demonstration of well-established antibodies such as CD45 and CD20 and enabled the demonstration of a wide range of new antibodies, such as CD8 and p53.

Numerous antigen retrieval solutions have been described, probably the most popular are 0.01 M sodium citrate buffer at pH 6.0 and 0.1 mM EDTA at pH 8.0. Although an expanding range of commercial antigen buffers at both high and low pH ranges is available, some are designed to improve the staining of specific antigens.

Most domestic microwave ovens are suitable for antigen retrieval and operate at 2.45 GHz, corresponding to a wavelength in vacuo of 12.2 cm ( Fig. 19.8 ). Uneven heating and the production of hot spots have been reported by some workers using the microwave oven. However, by using a volume of buffer between 400 and 600 ml in a suitably sized microwave-resistant plastic container, the problems of uneven heating may be minimized. A batch of up to 25 slides in a plastic staining rack can be irradiated at one time and accurate, even antigen retrieval achieved. The actual heating time will depend on the following factors:

• Wattage of the oven. Most domestic ovens use a magnetron with an output between 750 and 1000 W. An important point to remember is that the output of the magnetron will decrease with age and frequency of use. The magnetron should be checked for efficiency annually.

• Choice of antigen retrieval buffer.

• Volume of buffer being used.

• Fixation of the tissues under investigation, i.e. fixative used and duration of fixation. This is an important factor, although not as critical as when using proteolytic enzyme digestion. Tissue fixed for extended periods of time will require extended irradiation times. Conversely, poorly fixed tissues may require a reduction in the heating time.

• Thickness of the tissue section: 3 μm sections require less antigen retrieval than 5 μm sections.

• Antigen to be demonstrated. Certain nuclear antigens may require increased heating times.

If extended heating times are used with a small volume of buffer, the buffer may need topping up with distilled or de-ionized water. This should be performed halfway through the total heating duration. At no stage should the sections be allowed to dry out during the antigen retrieval process.

Pressure cooker antigen retrieval

suggested the use of a pressure cooker as an alternative to the microwave oven. By using a pressure cooker, showed that the batch variation and production of hot and cold spots in the microwave oven could be overcome. Pressure cooking is said to be more uniform than other heating methods. A pressure cooker at 15 psi (10.3 kPa) reaches a temperature of around 120°C at full pressure. It is this increased temperature which appears to be a major advantage when unmasking certain nuclear tissue antigens, e.g. bcl-6, p53, p21, estrogen receptor and progesterone receptor. The demonstration of these antigens can sometimes be weak when using microwave antigen retrieval.

It is preferable to use a stainless steel domestic pressure cooker, because aluminum pressure cookers are susceptible to corrosion from some of the antigen retrieval buffers ( Fig. 19.9 ). The pressure cooker should have a capacity of 4–5 liters, allowing a large batch of slides to be treated at the same time. As with the microwave oven, the use of charged microscope slides or strong adhesives such as Vectabond or APES is required to prevent section loss.

Steamer

Although quite a popular method in some parts of the world, steam heating appears to be less efficient than either microwave oven heating or pressure cooking ( ). Times in excess of 40 minutes are sometimes required, but the method does have the advantage of being less damaging to tissues than the other heating methods. Commercially available rice steamers are adequate for this purpose.

Water bath

demonstrated that a water bath set at 90°C was adequate for antigen retrieval. However, by increasing the temperature to 95–98°C, antigen retrieval was improved and the incubation times could be decreased. This technique has the advantage of being less damaging on the tissue sections because the temperature is set below boiling point. By using a lower temperature than other heating methods, the antigen retrieval buffer does not evaporate and expensive commercial antigen retrieval solutions can safely be reused. The method has the disadvantage that the antigen retrieval times are increased compared to other methods.

Advantages of heat pretreatment

Some antigens previously thought lost in routinely processed, paraffin wax embedded sections are now recovered by heat pretreatment. Many antigens are retrieved by uniform heating times, regardless of the length of fixation, e.g. up to several weeks in formal saline ( ). The demonstration of heavy-chain immunoglobulins is more reliable and reproducible than when proteolytic digestion is employed. The dilution factors of some primary antibodies ascertained with traditional methods can be increased when using heat pretreatment.

Pitfalls of heat pretreatment

Care should be taken to prevent the sections drying after heating, as this destroys antigenicity. The boiling of poorly fixed material also damages nuclear detail. Fibrous and fatty tissues tend to detach from the slide; this can sometimes be overcome by increasing the drying temperature to 56°C and using Superfrost Plus microscope slides. Alternatively, Vectabond or APES-coated slides can be dipped in 10% formal saline for 1–2 minutes and air dried before picking up the sections. This tends to improve the adhesion, probably by adding more aldehyde groups to the slide surface. Not all antigens are retrieved by heat pre-treatment, and the range of staining of some primary antibodies, e.g. PGP9.5, a neuroendocrine marker, is altered ( ).

Commercial antigen retrieval solutions

There are numerous commercial antigen retrieval solutions available. They can either be specialized high pH solutions, recommended for certain antibodies, or lower, pH 6.0 for more general use. These solutions may be a mixture of different chemicals, such as citrate and EDTA. They offer advantages over the ‘in house’ retrieval solutions as they are ready to use, require no pH calibration and are fully certified to comply with laboratory accreditation procedures. However, they can be expensive.

Enhancement and amplification

The optimum dilution of primary antibody for diagnostic IHC is defined as the concentration of the primary antibody which gives the optimal specific staining with the least amount of background staining. The optimal dilution will depend upon the type and duration of fixation. Serial dilutions of antibody will often give the distribution of reactivity shown in Fig. 19.10 :

• Poor reaction in area is due to steric hindrance of the labeling antibody accessing the primary antibody, the prozone effect. This is due to the primary antibody being too concentrated.

• Suboptimal reaction in area is caused by inadequate presence of primary antibody i.e. the primary antibody is too diluted.

The optimum concentration of primary antibody is that measured below the apex of the peak, and the use of several control sections with varying expression of antigen will aid the determination of a correct working dilution of primary antibody for each particular laboratory. Inter-laboratory variations in the choice of fixative, duration of fixation, paraffin wax processing, section treatment and the IHC detection system used, make the dilution of primary antibody unique to the laboratory which produced the paraffin wax section and accounts for the great variation in the dilution of primary antibodies used from laboratory to laboratory.

The dilutions of primary antibodies and labeling systems for diagnostic IHC should be ascertained on material where the antigen levels are adequate but not excessive. Occasionally situations arise where some tumors shed much of their antigen, e.g. prostatic tumors often express less prostate specific antigen (PSA) than normal prostatic glands. Enhancement and amplification by modification of demonstration techniques may be required to identify low levels of antigens. This can be achieved by the following methods:

• 1.

  Increasing the concentration of the primary antibody. This can usually be accomplished with most monoclonals without significantly increasing the background staining as this type of antibody, especially in the form of tissue culture supernatant, does not contain any non-specific contaminants. Polyclonal antibodies can give excessive background problems and it is advisable to use a casein blocking solution, as described in the methods later in this chapter. Occasionally the addition of a small amount of detergent, e.g. 0.01% Tween, to the washes helps to reduce background staining. Further details on dealing with background staining appear later in the text.

• 2.

  Prolonging incubation with the primary antibody overnight, at 4–8°C or at ambient temperature, can enhance staining. Many immunohistochemists employ this methodology for their routine work because higher dilution of primary reagents is achieved, allowing costs to be reduced. Dilutions must not be excessive, otherwise low levels of antigen will not be detected, resulting in false negative staining.

• 3.

  Increasing the concentration of the bridge reagent beyond the optimal dilution, or repeated application of the bridge reagent, marginally increases the sensitivity of the avidin-biotin systems. Furthermore, in the case of the CD15 primaries, which are IgM subclass antibodies, reported that an IgM link, as opposed to a broad-spectrum immunoglobulin bridge reagent, improves the rate of detecting CD15-positive Reed-Sternberg and Hodgkin cells ( Fig. 19.11 ). confirmed this work, but also indicated that when microwave antigen recovery was used in place of trypsin, further amplification was achieved.

  

• 4.

  Chemical enhancement of the reaction end product of the peroxidase-DAB reaction may be employed and can be achieved by the addition of imidazole ( ) or heavy metals such as copper or cobalt ( ).

• 5.

  Repeated applications of the bridge and label increase the sensitivity of the APAAP technique. Whilst the initial primary, bridge and label are incubated for 30 minutes each, the repeated applications of bridge and label require only 10 minutes each. After two such repeats enhancement is usually sufficient for most antibodies.

• 6.

  Changing the chromogen substrate, especially for alkaline phosphatase, gives a more intense reaction product. For example, nitro-blue tetrazolium is more intense than Fast Red and can be left on overnight to give probably the most intense reaction of all chromogens available today. The only drawback is that the blue-black reaction product does not contrast well with hematoxylin counterstaining. Improved commercial formulae of traditional substrates are superior to ‘in house’ formulae.

Multiple labeling techniques

The ability to label two or more different antigens in the same tissue section is playing a greater role in routine IHC ( Figs. 19.12 and 19.13 ). The use of automated IHC staining systems, a greater range of substrate chromogens and the use of commercially available double labeling kits has provided a much more robust method than was previously available. The user has the choice of using the same detection system with different substrates, or of using different detection systems for individual substrates.

The methodology involves sequential staining of each of the primary antibodies; practice will help the user determine the optimum order of labeling and the optimum substrates/chromogens to use. It is generally recognized that the primary antibody which gives the strongest final reaction product should be performed first (often with a DAB chromogen) followed by the weaker reaction. The choice of different enzyme-substrate combinations is important, as certain combinations work better and offer more contrast than others. The range of commercially available substrates from Vector Laboratories provides a wide choice. For example, when using alkaline phosphatase methods, the use of Vector Blue as the first substrate layer contrasts well when using Vector Red as the second substrate. For peroxidase methods the use of DAB as the first substrate contrasts well with Vector VIP as the second substrate.

Automation lends itself well to multiple labeling and this is facilitated by the availability of double labeling kits, e.g. Dako Envision™ DuoFLEX Doublestain system (HRP/AP) or the use of Leica Bond polymer Refine (DAB) with Bond Polymer Refine Red.

Fixation and paraffin wax block immunohistochemistry

A prerequisite for all routine histological and cytological investigations is to ensure preservation of tissue architecture and cell morphology by adequate and appropriate fixation (see Chapter 4 ). The most popular choice of fixatives for routine histology are formalin based, usually a 10% solution with the addition of phosphate buffers. The choice of fixative amongst pathologists was initially based on subsequent morphological appearance, and the clarity of established staining techniques using dyes which were the mainstay of diagnostic histopathology long before the advent of IHC. Most pathology teaching and learning is based on these traditional techniques, with all the artifacts they produce, and IHC has had to tailor itself to this type of material in order to become an effective diagnostic aid.

Prompt fixation of thin (3 mm) slices of tissue is essential to achieve consistent demonstration of tissue antigens. Delayed fixation or poor fixation may cause loss of antigenicity or diffusion of antigens into the surrounding tissue. Following fixation most material is routinely processed to paraffin wax to facilitate section cutting. It is therefore important to establish a fixation and processing procedure which retains good morphology and maximizes the ability of the immunohistochemist to identify the antigens which aid diagnosis. Ideally, if required, it should be possible, by prior arrangement with the surgeons and theater staff, to receive fresh specimens soon after surgery, in order that material can be selected for routine processing and frozen storage. The latter should be snap frozen using liquid nitrogen and stored at −80°C. This material can be used for preparing imprints for fluorescence in situ hybridization (FISH) techniques, as a source of RNA and DNA for molecular biology techniques, or for cutting frozen sections for the demonstration of antigens not readily demonstrated in paraffin wax sections. Recent advances have made it possible to perform some molecular techniques such as ISH on paraffin wax sections as well.

Retrospective studies are often hampered by a lack of knowledge of the duration of fixation. indicated that prolonged fixation reduces immunoreactivity and, with formalin fixation this tends to occur over a period of weeks rather than days. Certain antibodies such as CD20 and CD45 are less affected by fixation times. According to , microwave pretreatment enables the retrieval of antigens after formalin fixation even up to two years later. In general, the use of heat-mediated antigen retrieval has enabled a greater consistency of IHC staining over a wide range of different fixatives and fixation times ( Figs. 19.14 and 19.15 ). Most individual laboratories will employ a single fixative used over a range of fixation times between 18 and 72 hours, a single standardized heat methodology can often then be applied. Referral laboratories dealing with a wide range of material from different sources where the fixation regimes are unknown may need to use more than one retrieval method.

The advances in molecular technology enabled the production of duplicate synthetic peptide sequences which survive routine processing techniques. One such peptide was produced for the human CD3 antigen, fixed in a formalin fixative and subsequently used to raise a polyclonal antibody ( ). This reagent proved to be successful in the detection of T cells and related lymphomas in routinely processed paraffin wax sections. Using either proteolytic digestion or heat antigen retrieval, polyclonal anti-CD3 is an excellent example of how IHC has adapted to the prevailing conditions in histopathology.

Frozen sections

Despite the decline in the use of frozen sections for diagnostic purposes, IHC on frozen sections remains an important histological tool. Frozen sections have certain inherent disadvantages compared to paraffin wax sections, including poor morphology, limited retrospective studies and storage of material. The advantage of frozen sections is that the antigen is preserved and not cross-linked or hidden as can occur in paraffin wax sections.

The use of air-dried, unfixed cryostat sections of skin or renal biopsies allows the detection of chemically sensitive or otherwise labile antigens present in the tissue. In practice, unfixed cryostat sections or cell preparations are used whenever possible unless the antigen under investigation is known to be soluble, in which case suitable fixation such as cold 10% neutral buffered formalin can be used. Ethanol and acetone are other alternatives, but it should be noted that tissue morphology is sub-optimal with these methods.

Cytological preparations

The value of IHC on cytological preparations has been established. Acetone-fixed smears or cytospins are often preferred by the immunohistochemist, as this allows a wide range of primary antibodies to be employed without destroying the target epitopes. Many cytology laboratories still insist on fixing cytological preparations in alcohol as opposed to acetone, and consequently the number of demonstrable antigens may be limited, but the morphology may be superior. Where cytological samples are cellular enough, there is also the option of producing a cell block which can then be processed into paraffin wax. This gives greater flexibility for the number and range of tests which can then be performed on the sample and also provides archive material.

Automation

Following the introduction of heat antigen retrieval methods, there has been a continual increase in the numbers of antibodies effective on paraffin wax sections. Many of these antibodies can have a direct impact on diagnosis, prognosis or treatment. The move towards automation has also been driven by other forces, such as the need for greater standardization and consistency of results, a change in workforce, and the need to comply with national accreditation standards. Reflecting the current diagnostic turnaround targets there is now constant pressure to get a diagnostic result back to the clinician in as short a timeframe as possible. Many large diagnostic laboratories can be processing between 50-80,000 IHC slides per year. The use of automated systems not only increases productivity but also enhances quality assurance with full traceability of reagents. This is becoming more important for accreditation standards. Alongside automated staining systems many laboratories have also taken the step to move to pre-diluted antibodies. They are generally of good quality and produce consistent, reliable staining, as well as saving time and reducing errors e.g. less pipetting.

The selection of a suitable autostainer may be influenced by many factors but should take into account aspects such as:

• Capacity

• Flexibility

• Reliability

• Ease of use

• Cost per slide.

The more recently introduced autostainers, e.g. the Dako OMNIS and Ventana Benchmark Ultra, allow a LEAN workflow pattern, moving away from batch processing to a more continual loading of slides. This allows greater flexibility and makes prioritization of cases much easier.

Wherever possible, it is recommended to trial the different autostainer options, and assess them with the laboratory’s own material and their routine antigen panels. Prioritization of the automated immunostainer criteria will differ between diagnostic and research facilities. The options have previously been outright purchase, lease of the equipment and/or a reagent rental contract. Most diagnostic laboratories would normally go with a reagent rental contract now, particularly as they may need multiple instruments to fulfill their capacity requirements. Each method has its advantages and disadvantages. Purchase involves one payment but does not include maintenance after the initial warranty or replacement costs. Leasing of the equipment or a reagent rental contract will cover the maintenance costs and equipment is replaced as required.