18 Immunohistochemical techniques
Introduction
In 1941 Albert H. Coons described a revolutionary new way of visualizing tissue constituents using an antibody labeled with a fluorescent dye. Visualization of the labeled complex was achieved by the use of a fluorescence microscope. The first fluorescent dye to be attached to an antibody was fluorescein isocyanate, but fluorescein isothiocyanate soon became the label of choice because the molecule was much easier to conjugate to the antibody and the result was more stable (Riggs et al. 1958). Fluorescein compounds emit a bright green fluorescence when excited at a wavelength of 490 nm. The technique has enormously expanded and developed following the early work. New labels have been introduced, including red, yellow, and blue fluorochromes. This permits the simultaneous visualization of several separately labeled antibodies on a single preparation. Currently, fluorescein isothiocyanate and rhodamine are among the most popular fluorochromes. This methodology, whilst useful in some diagnostic areas, such as determining the nature of protein deposits in skin and renal diseases, and bacteria in infected material, has certain limitations. A fluorescence microscope is necessary to visualize the fluorochrome, and this has a tendency to fade. More importantly and probably the greatest disadvantage of immunofluorescence is that it is difficult to demonstrate the morphological detail of the labeled cell and the associated tissue components. The success of immunohistochemistry in some areas of pathology stimulated interest in the development of alternative antibody labeling techniques that would avoid the difficulties and limitations associated with immunofluorescence.
Many limitations were overcome with the introduction of enzymes as labels. Cells that have been labeled with an enzyme such as horseradish peroxidase, conjugated to an antibody, and visualized with an appropriate chromogen such as diaminobenzidine (DAB) (Nakane & Pierce 1966), can be counterstained with traditional nuclear stains such as hematoxylin. This permits the simultaneous evaluation of both specific immunohistochemistry and morphological detail. In 1970 Sternberger et al. described the peroxidase-anti-peroxidase (PAP) technique. In 1971, Engvall and Perlman reported the use of alkaline phosphatase labeling, and Cordell et al. (1984) described the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique. Heggeness and Ash (1977) proposed the use of avidin-biotin for immunofluorescence. This technique was modified by Guesden et al. (1979) and Hsu et al. (1981) who used a horseradish peroxidase label. Avidin-biotin labeling was superseded by streptavidin-biotin labeling, and was one of the more popular techniques used in diagnostic laboratories. However, labeled polymer detection systems are now the most popular choice for most diagnostic laboratories.
Certain epitopes, for example the proliferation antigen, Ki67 and T-cell antigens CD2, CD4, CD5 (Fig. 18.2), CD7 and CD8, can only be demonstrated in formalin-fixed, paraffin wax-processed tissue after heat pretreatment, and then only with certain monoclonal antibody clones. Antibodies such as those directed against the leukocyte common antigen (clones PD7/2B11) and the CD20 antigen (clone L26) produce enhanced staining after citrate buffer (pH 6.0) heating. Surprisingly, heat pretreatment allows for considerably greater dilution factors. The demonstration of antigens such as cyclin D1 (with clone DCS-6), on the other hand, is better in a high pH solution (Tris-EDTA, pH 10.0). However, the use of the cyclin D1 rabbit monoclonal antibody (clone SP4) produces very good results using citrate buffer, pH 6.0 (Fig. 18.3).
Immunohistochemistry theory
Definitions
Production of primary reagents
Monoclonal antibodies
The development of the hybridoma technique by Kohler and Milstein in 1975 to produce monoclonal antibodies has revolutionized immunohistochemistry by increasing enormously the range, quality, and quantity of specific antisera. Detailed descriptions of the technique have been given by Gatter et al. (1984) and Ritter (1986).
This approach to the production of monoclonals has dramatically increased the number of antibodies available for immunohistochemistry and has allowed for further evolution with the ability to identify more antigens in paraffin sections. Detailed comparisons of the values and limitations of polyclonal and monoclonal antibodies have been given by Warnke et al. (1983) and Gatter et al. (1984).
Lectins
Lectins are plant or animal proteins that can bind to tissue carbohydrates with a high degree of specificity according to the lectin and the carbohydrate group (Brooks et al. 1996). Since the carbohydrates may be characteristic of a particular tissue, lectin binding may have diagnostic significance (Damjanov 1987). They can be labeled in similar ways to antibodies, or identified by using lectin-specific antibodies as secondary reagents (Leatham 1986).
Labels
Enzyme labels
Horseradish peroxidase is the most widely used enzyme, and in combination with the most favored chromogen, i.e. 3,3α-diaminobenzidene tetrahydrochloride (DAB), it yields a crisp, insoluble, stable, dark brown reaction end-product (Graham & Karnovsky 1966). Although DAB has been reported to be a potential carcinogen, the risk is now thought to be low (Weisburger et al. 1978).
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.
Other chromogens are available, including: 3-amino-9-ethylcarbazole (Graham et al. 1965; Kaplow 1975), which gives a red final reaction product; 4-chloro-1-naphthol (Nakane 1968), producing a blue final reaction product; Hanker-Yates reagent (Hanker et al. 1977), producing a dark blue product; and α-naphthol pyronin (Taylor & Burns 1974), which produces a red-purple final reaction product.
Colloidal metal labels
When used alone, colloidal gold conjugates appear pink when viewed using the light microscope. A silver precipitation reaction can be used to amplify the visibility of the gold conjugates (Holgate et al. 1983a). In addition, both gold and silver-enhanced gold conjugates can be visually emphasized using polarized incident light (epi-illumination) microscopy (Ellis et al. 1988) (Fig. 18.4a, b).
Immunohistochemical methods
Two-step indirect technique
Unlabeled antibody-enzyme complex techniques (PAP and APAAP)
The original immunoenzyme bridge method using enzyme-specific antibody (Mason et al. 1969; Sternberger 1969) was rapidly superseded by an improved version using a soluble peroxidase-anti-peroxidase complex (PAP) (Sternberger et al. 1970). These complexes are formed from three peroxidase molecules and two anti-peroxidase antibodies (Fig. 18.5d), and are used as a third layer in the staining method. They are bound to the unconjugated primary antibody, e.g. rabbit anti-human IgG, by a second layer of ‘bridging’ antibody that is usually a swine anti-rabbit applied in excess so that one of its two identical binding sites binds to the primary antibody and the other to the rabbit PAP complex.
Immunogold silver staining technique (IGSS)
The use of colloidal gold as a label for immunohistochemistry was introduced by Faulk and Taylor (1971). It can be used in both direct and indirect methods and has found wide usage in ultrastructural immunolocalization. It is not widely used in light microscope immunohistochemistry even after the advantages of silver development reported by Holgate et al. (1983a). In this method the gold particles are enhanced by the addition of metallic silver layers (Fig. 18.5e) to produce a metallic silver precipitate which overlays the colloidal gold marker and which can be seen with the light microscope. The technique uses silver lactate as the ion supplier and hydroquinone as the reducing agent in a protective colloid of gum arabic at pH 3.5. In some instances section pretreatment with Lugol’s iodine and sodium after dewaxing and rehydration may be required to improve staining intensity. The method is generally accepted to be more sensitive than the PAP technique, but suffers from the formation of fine silver deposits in the background, especially in inexperienced hands. It can be confusing when trying to identify small amounts of antigen. Modifications to the original technique have been reviewed by De Mey et al. (1986).
(Strept) avidin-biotin techniques
Streptavidin can be isolated from the bacterium Streptomyces avidini, and like avidin it has four high-affinity binding sites for biotin. However, in practice due to the molecular arrangement of these binding sites, fewer than four biotin molecules actually bind. Biotin (vitamin H) is easily conjugated to antibodies and enzyme markers. Up to 150 biotin molecules can be attached to one antibody molecule, often with the aid of spacer arms. By spacing the biotins, the large streptavidin has room to bind and maximize its strong affinity for biotin. The streptavidin-biotin technique can employ either enzyme label bound directly to the streptavidin (Guesden et al. 1979); alternatively, the enzymes are biotinylated and the biotinylated label, forming the streptavidin-biotin complex (Hsu et al. 1981), occupies 75% of the streptavidin-binding sites. Usually the latter is commercially supplied as two separate reagents, biotinylated label and streptavidin, and they are added together 30 minutes before use in order for the complex to form fully. Careful stoichiometric control ensures that some binding sites remain free to bind with the biotinylated secondary antibody. As a large number of biotins can be attached to a single antibody, then numerous labeled streptavidin molecules may be bound on top. This produces increased sensitivity compared to the previously described enzyme techniques and allows a higher dilution of the primary antibody. Tissues rich in endogenous biotin such as liver and kidney will require the use of an avidin/biotin block before applying the primary antibody.
Hapten labeling technique
Bridging techniques using haptens such as dinitro-phenol and arsanilic acid have been advocated (Jasani et al. 1981, 1992). In this technique, the hapten is linked to the primary antibody and a complex is built up using an anti-hapten antibody and either hapten-labeled enzyme or hapten-labeled PAP complex.
Biotinylated tyramide signal amplification
Bobrow et al. first described the use of biotinylated tyramide to enhance signal amplification, in 1989. Subsequent work by Adams in 1992 and King et al. in 1997 enabled the development of a highly sensitive detection system. In conjunction with heat-induced epitope retrieval techniques, the use of biotinylated tyramide amplification enabled many antigens which had previously been unreactive in formalin-fixed paraffin-embedded tissue to be demonstrated. Antibodies could be used at far greater dilutions than in conventional techniques. The biotinylated tyramide amplification reagent was first available commercially from DuPont; this was subsequently followed by the CSA (Catalyzed Signal Amplification) kit from Dako.
Unmasking of antigen sites
The concept that antigens can be masked by the chemical processes involved in formalin fixation and paraffin processing and that some form of unmasking of these antigens is required dates far back into the history of immunohistochemistry (Brandtzaeg 1983). The majority of antigen unmasking studies have been applied to formalin-fixed material. When formalin-based fixatives are used, intermolecular and intramolecular cross-linkages are formed with certain structural proteins. These are responsible for the masking of the tissue antigens. This adverse effect has been thought to be the result of the formation of methylene bridges between reactive sites on tissue proteins (Bell et al. 1987; Mason & O’Leary 1991). These reactive sites include primary amines, amide groups, thiols, alcoholic hydroxyl groups, and cyclic aromatic rings. The degree of masking of the antigenic sites depends upon the length of time in fixative, temperature, concentration of fixative, and availability of other nearby proteins able to undergo cross-linkage.
Manual methods for antigen unmasking include:
• Proteolytic enzyme digestion
• Combined microwave oven irradiation and proteolytic enzyme digestion
• Pressure cooker inside a microwave oven
Proteolytic enzyme digestion
Pretreating formalin-fixed routinely processed paraffin sections with proteolytic enzymes to unmask certain antigenic determinants was described by Huang et al. (1976), Curran and Gregory (1977), and Mepham et al. (1979). The most popular enzymes employed today are trypsin and protease, but other proteolytic enzymes 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. Nevertheless, it is generally accepted that the digestion breaks down formalin cross-linking and hence the antigenic sites for a number of antibodies are uncovered.
Heat-mediated antigen retrieval techniques
Another possible theory was described by Morgan et al. (1997), who postulated that calcium coordination complexes formed during formalin fixation prevent antibodies from combining with epitopes on tissue-bound antigens. The underlying theory of calcium involvement is that hydroxymethyl groups and other unreacted oxygen-rich groups (e.g. carboxyl or phosphoryl groups) can interact with calcium ions to produce large coordinate complexes which can mask epitopic sites by steric hindrance. The high temperature weakens or breaks some of the calcium coordinate bonds, but the effect is reversible on cooling, because the calcium complex remains in its original position. The presence of a competing chelating agent at the particular temperature at which the coordinate bonds are disrupted removes the calcium complexes. Evidence to support this theory comes from the chemical nature of some of the antigen retrieval reagents, such as citrate buffer and EDTA. In addition it has been shown that the inclusion of calcium ions with an unmasking reagent inhibits its effectiveness (Morgan et al. 1994).
Microwave antigen retrieval
Shi et al. (1991) first established the use of microwave heating for antigen retrieval. However, the use of heavy metal salts posed a significant risk to the health and safety of the users. Gerdes et al. (1992) used microwave antigen retrieval with a non-toxic citrate buffer at pH 6.0 and demonstrated the Ki67 antigen, which had previously thought to be lost during formalin fixation and paraffin processing. The results were equivalent to those seen in frozen sections. Cattoretti et al. (1993) 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.
• 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, in terms of 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.
Pressure cooker antigen retrieval
Norton et al. (1994) suggested the use of the pressure cooker as an alternative to the microwave oven. By using the pressure cooker, Norton et al. (1994) provided evidence 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 that appears to be a major advantage when unmasking certain nuclear tissue antigens such as bcl-6, p53, p21, estrogen receptor, and progesterone receptor. The demonstration of these antigens can sometimes be weak when using microwave antigen retrieval.
Combined microwave antigen retrieval and trypsin digestion
Detection of low levels of antigen
Enhancement and amplification
1. Increasing the concentration of the primary antibody. Usually this can be accomplished with most monoclonals without increasing the background staining significantly, 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. Sometimes 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 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 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, LeBrun et al. (1992) 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. 18.9). Charalambous et al. not only confirmed this work in 1993, but they 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-diaminobenzidine method can be achieved by the addition of imidazole (Straus 1982), heavy metals such as copper or cobalt (Hsu & Soban 1982), osmication, or treatment with gold chloride. Colloidal gold labeling can be enhanced dramatically using silver salts with the IGSS technique (Holgate et al. 1983b).
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 used. Some chromogen substrate solutions, especially for alkaline phosphatase, give a more intense reaction product than other reagents. For example nitro-blue tetrazolium is not only more intense than fast red but also 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.
7. Techniques for elevating the sensitivity of the extended polymer-labeled antibodies and other pre-diluted reagents are more restricted. The disadvantage of pre-diluted antibodies is that the dilutions selected are not necessarily suitable for the multitude of fixation and processing protocols employed. Hence, weak staining can only be overcome by increasing the incubation times, elevating the temperature to 37°C, or chemical enhancement of the diaminobenzidine reaction product, or other appropriate substrate.
8. Tyramide signal amplification. In 1989, Bobrow and fellow workers described a novel signal amplification method, catalyzed reporter deposition, and its application to immunoassays. In 1992 the same group proposed that this method would be suitable for immunohistochemistry. Erber et al. and King et al. in 1997 quite independently published data showing that this novel signal amplification system employed with avidin-biotin systems showed greater sensitivity than that provided by the more conventional avidin-biotin methods.