Immunohistochemistry (IHC) is a powerful tool that is used in diagnostic practice and research . It is used in the search for cell or tissue antigens ranging from amino acids and proteins to infectious agents and specific cellular populations . Brandtzaeg reported that immunostaining for cell markers represents a means to ‘talk with cells’, because it allows not only the histological origin of the cell to be identified but also indicates its function in vivo, when properly examined with the correct antibodies.
The immunohistochemical reactions can be used in different situations. The most important are histogenetic diagnosis of morphologically nondifferentiated neoplasias; subtyping of neoplasias (such as lymphomas); characterization of the primary site of malignant neoplasias; research for prognostic factors and therapeutic indications of some diseases; discrimination of benign versus the malignant nature of certain cell proliferations; and identification of the structures, organisms, and materials secreted by cells .
During the last decade, there has been a marked increase in publications on IHC. A search in the Medline database of PubMed using the key word immunohistochemistry from the year 2000 to 2010 showed an increasing number of publications every year (Fig. 1). This indicates the importance of this technique as a tool in scientific research and in the differential diagnosis of many clinical diseases that cannot be determined by conventional analysis using H&E.
History of immunohistochemistry
The history of IHC dates back to 1941, when the antibody was labeled with a fluorescent dye and used to identify an antigen in tissue sections. Since the 1970s, the use of IHC techniques has increased exponentially, in parallel with the development of specific molecular markers. The discovery of antigen retrieval (AR) methods (exposure of antigen epitopes present in the tissue, favoring the antigen–antibody reactions for the next stages of the technique) has resulted in some degree of consistency, allowing IHC to be used reliably as a diagnostic tool. The systems of secondary antibody detection also allowed IHC to be used in fresh specimens as well as in fixed tissues . This led to further acceptance of IHC as an invaluable technique.
The need for immunohistochemistry standardization
Standardization of IHC requires optimal conditions (e.g. incubation time, temperature, dilutions, controls, detection system, etc.) to ensure the selectivity of an antibody reaction to the expected antigen. The main aim of standardization in IHC is to obtain reproducible and consistent results within each laboratory and comparable results among different laboratories. In clinical applications, this may translate into increased diagnostic accuracy and improved patient care because therapies are now being directed toward molecular targets .
Despite the widespread use of IHC, there is a lack of standardization among different laboratories. Immunostaining has three stages: preanalytical, analytical, and postanalytical. Each stage has many variables that influence the demonstrability of tissue antigens and can consequently affect the reliability of the stain (Fig. 2). This myriad of variables does not allow meaningful comparisons of results obtained in different laboratories and even in the same laboratory . Therefore, it is important to highlight tissue processing and staining variables that may alter the results of IHC and assess the magnitude of reported factors in the literature that require standardization.
Variables affecting the reliability of the immunohistochemistry stain Preanalytical variables
They include tissue preservation, fixation, and tissue processing. Many of the preanalytical variables have a major influence on antigen preservation .
Processing of tissue for the analysis of IHC begins with obtaining the tissue sample. The size of the sample itself is an important factor. If the sample is small, the tissue will be uniformly well fixed rapidly. In large samples, heterogeneity in the IHC stain is detected probably because of a delay in the fixation of the cells in the center compared with the cells in the periphery of the sample . Several manipulations of the tissues such as immersion in distilled water or drying the sample in air for 18–24 h before fixation can also alter the results of the stain .
Fixation of the tissue
Fixation of tissues is an important step to arrest autolysis and putrefaction, coagulate soluble and structural proteins, and facilitate staining . Although some authors have reported that a delay in fixation has no effect on the IHC results , many other pathologists have confirmed that delayed fixation can affect IHC staining negatively. They recommend immediate fixation of the sample and limiting prefixation delays to 1h or less [7,10].
Fixation of tissues can be carried out by formalin to form cross-links in tissue, alcohol, and acetone to coagulate and dehydrate tissues, cryopreservation to inactivate endogenous enzymes, and microwave energy to thermally denature enzymes and fix the tissue . To date, there is no ‘one fixative that can fit for all’ in IHC. Cryopreservation provides good results of IHC but with a relatively poor morphology and an insufficient definitive pathologic diagnosis . Frozen tissues also require large and expensive storage facilities, which make their use prohibitive for most medical practices and limit their use for a molecular diagnosis. However, formalin fixation and paraffin embedding are applied in over 90% of cases in hospitals and clinical settings as they provide superior morphological details and high consistency under various conditions, and are simple and allow economical processing and handling .
The type of fixative used in tissue fixation for IHC is an important factor. Many researchers recommend the use of 10% neutral-buffered formalin (NBF) as a standard fixative in IHC [7,10]. However, this may not be appropriate in all immunostaining procedure. Some authors have reported that the intensity of epidermal growth factor receptor immunostaining is superior in specimens preserved with 4% unbuffered formalin compared with 10% unbuffered formalin or 10% NBF . The pH and the concentration of the formalin solution used for specimen fixation also affect the quality of immunostaining. Most of the antigens yielded consistent immunostaining and superior preservation when specimens were preserved in 10% NBF at pH 5–7 compared with unbuffered formalin or NBF at a lower or a higher pH .
The buffer used for NBF is also a factor that may affect the quality of immunostaining. Although PBS has been reported to be the preferred buffer , T-lymphocyte surface membrane clusters of differentiation (CD) antigens CD3, CD4, and CD8 showed superior intensity and reduced background staining when specimens were preserved in 10% formalin buffered in Tris solution compared with PBS .
Other fixatives such as alcoholic fixatives have been used primarily to avoid the loss of antigenicity caused by excessive formalin fixation or for monoclonal antibodies reacting against an epitope destroyed by formalin. These fixatives are typically applied when examining lymphocytes using CD-specific markers and when examining immunoglobulins such as IgG, A, and M .
Duration of fixation
The optimum duration of fixation is a point of interest in many literatures because of conflicting results. Some laboratories have estimated the minimum time required for fixation as 6–8h , whereas the long duration of fixation may extend to weeks . Overfixation for a long duration has been associated with reductions in the intensity and the extent of immunostaining (Fig. 3a and b), whereas underfixed specimens have shown graded staining from the periphery to the core because of tissue necrosis . Definitive thresholds for stable immunostaining remain undefined. In many publications, tissue fixation in formalin for 24h has been reported to be the most reliable for several antigens [14,20,21].
Published guidelines for IHC recommend that specimens be subjected to 1–15h of dehydration, 15min–3h per alcohol stage with five recommended stages, with the duration depending on the type and the size of the specimen. Tissue processing in paraffin at high temperatures (over 60°C) may compromise specimen antigenicity. After tissue sectioning, drying of slides at room temperature for 24h or at 50–60°C for a minimum of 1h is also recommended . The block should preferably be sectioned at a thickness ranging between 3 and 7μm on slides prepared previously with some kind of adhesive (such as silane and polylysine). Slices less than 3μm in thickness could result in very weak immunostaining, whereas those more than 7μm in thickness may lead to loss of tissue on the glass slide or may alter the results of the immunostaining analysis .
Other techniques for tissue processing
Alternative processing systems include the application of ultrasound or microwaves. Ultrasound, per se, does not preserve tissue, but is often used in combination with fixatives to increase tissue penetration markedly and, consequently, the speed of fixation .
Microwave-assisted tissue processing can also provide a reliable alternative to conventional tissue processing in the diagnostic surgical pathology laboratory, as the immunohistochemical results obtained on tissue processed by microwave are comparable in quality to that performed on tissue processed using conventional methods . The time required for sufficient tissue processing may be reduced when performed in conjunction with microwave irradiation or ultrasound sonication. However, published recommendations are not yet available on the applications of such techniques as a standard .
These include AR, primary antibodies, detection systems (secondary antibodies), and the use of tissue controls .
AR is the term used to describe the restoration of the antigen's optimal immune reactivity after formalin fixation. For the majority of tissue antigens, formalin fixation will lead either to a reduction in their functional affinity for the antibody (avidity) or will completely erase the antigen's intrinsic affinity. The measurable effect that formalin fixation had on most tissue antigens was either reduced or absent staining, respectively. Formalin fixation reduces the electrostatic charges necessary for attracting and reacting with the antibody. Furthermore, formalin fixation modifies the tertiary structure of proteins in the antigens, making them undetectable by specific antibodies sometimes . Also, multiple bonds are formed among proteins in the tissue sections by the establishment of resistant methylene bridges between protein end-groups. Such methylene cross-links can mask the antigenic epitopes to be detected with the immune reaction .
One of the challenges in IHC is to develop methods that reverse the changes produced during formalin fixation. Without AR, the immune reaction of the target antibody will be masked, resulting in a false-negative reaction (Fig. 3c). The exact mechanism by which AR works on formalin-fixed tissues is not clear. A variety of pathways may contribute to its success, including breakage of cross-linkages, extraction of diffusible blocking proteins, precipitation of proteins, calcium chelation, paraffin removal, and rehydration of tissue, resulting in better penetration of antibody and increased accessibility to antigen .
The most common procedures for AR used in IHC are heat based and enzymatic retrieval. Heat-induced AR continues to be the most effective method for the observation of a vast range of antigens in formalin-fixed tissues. The relationship between temperature and exposure time is inverse: the higher the temperature, the shorter the time needed to achieve beneficial results. The pH of the retrieval solution is also important. A low pH buffer (acetate, pH 1.0–2.0) is useful for nuclear antigens. The common buffers used in heat-induced AR are 0.01mol/l citrate buffer (pH 6.0), 0.1mol/l Tris-HCl (pH 8.0), and 1mmol/l EDTA–NaOH solution (pH 8.0) .
Enzymes are considered to digest the tissue to some degree, allowing antibodies to recognize antigenic sites. The commonly used enzymes include trypsin, proteinase K, pepsin, pronase E, and ficin. A combination of heat and protein digestion may be necessary to observe some antigens .
Other methods of AR have also been reported such as ultrasound fixation, which substantially increases protein antigenic reactivity in IHC staining .
Definite steps are performed in the immunostaining process including the application of a primary antibody specific for the target protein and visualization of the immune reaction by a specific detection system. Many reagents are used during the immunostaining process. The knowledge of each reagent's characteristics requires new titration in each new batch or clone. Selection of the proper dilution yields the best results .
Types of primary antibodies
Primary antibodies comprise two categories: polyclonal and monoclonal. The polyclonal antibodies result from animal immunization (example: rabbit, goat, monkey, rat, mouse, ewe, etc.). They can recognize many epitopes of the same antigen with high affinity and wide reactivity but lower specificity when compared with monoclonal antibodies. The monoclonal type, however, is developed from hybrids and provides antibodies against only one antigen epitope, yielding more specific results. This specificity reduces (although does not completely remove) the possibility of cross-reactivity with other antigens . Among the commercial types, companies specializing in the production of antibodies commonly provide important information about the format of the antibodies (e.g. purified, whole serum), the host in which the antibody was produced (e.g. mouse, rabbit), the protein concentration, the immunogen used (including epitope and molecular mass, if known), species reactivity (e.g. to human, mouse, others), cellular localization (e.g. cytoplasmic, membrane, nuclear), positive tissue control recommended, application of the antibody (e.g. immunoprecipitation, western blotting, enzyme-linked immunosorbent assay, immunohistology–formalin/paraffin, and frozen), and the relevant literature. It is advisable to contact the manufacturer for additional information on species cross-reactivity not indicated in the catalog or cross-reactivity with other antigens (e.g. serotypes of viruses, other related viruses or bacteria, cell antigens) that might result on fixation or AR .
A proper dilution of the primary antibody will contribute to the quality of the immunostaining. Manufacturing companies may offer ready-to-use reagents or recommend a special dilution on the basis of different researches published. Antibodies are diluted using different buffers. The concentration of ions in the buffer is a critical factor. Many authors have considered 0.05–0.1mol/l Tris buffer (pH 6.0) to be the most suitable diluent buffer for both monoclonal and polyclonal antibodies . PBS is also a widely used antibody diluent that may exert one of the most pronounced negative effects on staining intensities because this diluent contains Na+ ions from NaCl. Varying the concentration of Na+ ions (in the form of NaCl) can markedly influence the staining intensities at all antibody concentrations. They can form a ‘shield’ around the negatively charged antigens, thereby reducing or obstructing the attraction of the positively charged antibodies . Almost all monoclonal antibodies stain more intensely in the absence of NaCl .
Detection systems (secondary antibodies)
The antigen–antibody detection approach includes direct and indirect methods. In the direct method, the primary antibody is conjugated to a reporter molecule or a label that could be an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), which is then activated by adding a substrate or is conjugated to a fluorophore for detection by fluorescence microscopy. This method is quick but lacks sufficient sensitivity. The indirect method uses a secondary antibody with specificity against the unlabeled primary antibody to amplify the primary signal. After multiple secondary antibodies bind to each primary antibody, the enzyme label (usually horseradish peroxidase or AP) conjugates with the substrate to yield a chromogenic response. A further advantage of using enzyme-labeled systems is that the product can be made electron dense for electron microscopy .
Different methods can be used for the detection of the secondary antibody. The avidin–biotin–peroxidase complex and the labeled streptavidin–biotin complex are the most commonly used detection systems. However, specific situations, in which some tissues such as the liver and kidney contain endogenous biotin or avidin, may require the use of alternative detection methods to avoid high background staining . Polymer-based immunohistochemical methods do not rely on biotin. Therefore, they are gaining popularity in staining tissue with endogenous biotin. However, they are expensive . The tyramide amplification methods are another detection system that have increased sensitivity by at least 50-fold or greater than conventional methods, making it the one of choice in cases where antigens are present in very low amounts in the tissue .
Tissue controls in immunohistochemistry
The IHC technique is highly variable across different laboratories and includes many factors that can affect the final results. Therefore, the use of tissue controls during the staining process is important to verify the accuracy of the staining results .
Positive tissue controls: A positive control slide is a tissue known to contain the antigen under study. It should be prepared in the same manner as the samples examined and should be included for each set of tests. Ideally, this control should contain a spectrum of weak to strongly positive reactivity. A tissue with weak positive staining is more suitable than a tissue with strong positive staining for optimal quality control and to detect minor levels of reagent degradation. If positive tissue controls do not perform as expected, the results of the test should be considered invalid .
Negative tissue controls: A negative control is a tissue that should not contain the specific antigen to be tested, or control tissue obtained by excluding the primary antibody, or using normal serum or an unreactive antibody instead. Tissue used for negative controls should be prepared in the same manner as the tissue sample. If positive staining occurs in the negative tissue control, the results of the staining should be considered invalid .
Internal tissue controls: Internal controls, also known as ‘built-in’ or intrinsic positive controls, contain the target antigen within normal tissue elements, in addition to the tissue elements to be evaluated. Thus, they can replace external positive controls. This is ideal, as the tissue elements to be evaluated have been treated exactly as the internal control. An example of an internal control is desmin, which is present in blood vessel musculature , or endothelial nitric oxide synthase, expressed in the endothelium lining blood vessels (Fig. 3d). If the control slide does not produce the expected reaction, the staining must be repeated under the standard conditions until acceptable reactivity of control tissue is achieved.
Cell line controls: Some manufacturing companies sell cell lines separately and they can be used as a part of the diagnostic kits. They are developed specifically to monitor staining of the antigen of interest, and should be included in all stain runs as an additional protocol control. Cell line controls may be positive or negative. An ideal negative cell line control will contain a certain amount of target antigen, sufficiently low to produce no staining if the procedure has been performed correctly. At the same time, the amount should be sufficiently high to produce a weakly positive stain if the run has been performed under conditions that produce an excessively strong stain. An ideal positive cell line control would contain a number of target antigens producing both stains that are too weak and stains that are too strong [36,38].
This is one of the most common problems in IHC that results from different causes. Fixation of the tissue in formalin renders the protein more hydrophobic as a result of cross-linking between reactive amino acid. The increased hydrophobicity of proteins increases the background staining in IHC; therefore, prolonged fixation in formalin or other aldehyde-based fixatives should be avoided . The most common method to reduce background from hydrophobic interactions is the use of blocking protein before incubation of the primary antibody. This protein must be of the type that competes effectively for the hydrophobic binding sites in the tissues such as BSA .
In the enzyme-based detection systems, the presence of such enzymes, endogenously, in the tissues can produce a nonspecific reaction. Endogenous peroxidase activity in some tissues such as red blood cells can react with 3,3-diaminobenzidine, which is the chromogenic substrate for peroxidase, to produce a brown product resembling the positive immune reaction (Fig. 3e and f). Endogenous peroxidase is almost destroyed during formalin fixation. Pretreatment of tissue sections with a diluted solution of H2O2 will completely abolish any remaining peroxidase activity . AP can be used as a reporter in enzyme-based detection systems. However, endogenous AP can produce a nonspecific reaction. Two isoenzymes of AP are present in mammalian tissues: intestinal and nonintestinal forms. The nonintestinal form is easily inhibited by levamisol, whereas the intestinal isoform can be blocked with 1% acetic acid . Endogenous avidin could result in nonspecific binding, producing background staining. This nonimmune binding can be prevented either by preparing the solutions at pH 9.4 instead of at 7.6 or by using streptavidin instead of avidin, which can reduce the nonspecific binding significantly . Endogenous biotin is widely dispersed in mammalian tissues, particularly in the liver, lung, spleen, adipose tissue, mammary gland, kidney, and brain. Background from endogenous biotin is markedly decreased after formalin fixation but can be pronounced in frozen sections. Harsh heat-based AR methods expose endogenous biotin in formalin-fixed tissues. Binding of avidin used in detection systems to endogenous biotin can produce a strong background and needs to be inhibited. This can be suppressed with alkaline buffers, preincubation of tissue sections with unlabeled avidin and biotin, or incubation with nonfat dry milk .
Cross-reactivity can be a source of a nonspecific IHC stain that may lead to a false-positive stain, indicating that the molecule is expressed in that tissue, whereas, in reality, it is not. This occurs when the epitope of the antigen is similar to the epitope of completely unrelated molecules, because of a combination of binding affinity and structural homology, resulting in high-affinity binding of the primary antibody to an unrelated and unintended molecule. Cross-reactivity can be detected in polyclonal rather than monoclonal antibodies .
Antigen diffusion can cause background staining when the antigen is displaced from its original site. Optimal fixation can decrease the antigen diffusion as it anchors the antigen in place. However, mechanical injury, necrosis, or dryness of the tissue before fixation can show excess background staining .
Postanalytical variables range from the method of interpretation and description of the IHC staining results up to the accreditation of the laboratory itself. The results of the immunohistochemical stain test have no common quantitative measures. Instead, the results are typically based on subjective interpretation by microscopists with varying experience. Nowadays, the contradictory results reported in the literature are because of the lack of a definition on what constitutes a positive result and what does not. The use of computer-based assessments programs and semicomputerized programs is recommended in the interpretation of immunostaining results. Quality control and assurance programs are also crucial and should be focused on by both manufacturers and laboratory users. A number of scientific bodies have quality programs or quality assessment services. These programs should be considered as an aid or as a means of external assistance and should never replace national requirements for internal quality control .
As immunohistochemical techniques continue to evolve, their application in research and in pathology diagnosis is becoming increasingly valuable. Despite this, there is no standard method that can be applied for immunohistochemical analysis. Specimens have different preanalytical, analytical, and postanalytical variables and methods of interpretations that may affect the results. Standardization of such variables will provide more reliable and trustable results. Therefore, standardization of the IHC technique is required in clinical and research applications.
Conflicts of interest
There is no conflict of interest to declare.
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