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 Table of Contents  
Year : 2015  |  Volume : 1  |  Issue : 4  |  Page : 115-122

An Update on Immunohistochemistry in Translational Cancer Research

Department of Chemistry and Biochemistry, Harper Cancer Research Institute, University of Notre Dame, Notre Dame, IN, USA

Date of Submission08-May-2015
Date of Acceptance02-Aug-2015
Date of Web Publication27-Aug-2015

Correspondence Address:
Dr. Zonggao Shi
Department of Chemistry and Biochemistry, Harper Cancer Research Institute, University of Notre Dame, 1234 Notre Dame Avenue, A229 Harper Hall, South Bend, IN 46617
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2395-3977.163802

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Immunohistochemistry (IHC) takes advantage of the specific binding between antigen and antibody to measure the presence and abundance of antigen while simultaneously providing morphologic context on a tissue section. Since the revolutionary application of heat-induced epitope retrieval methods on formalin-fixed paraffin-embedded tissues, which started in early 1990s, IHC has been routinely used in diagnostic pathology. This approach has also enabled mining of the rich archives of pathologic specimens for exploration in translational cancer research. Newer IHC biomarkers are being continuously found as aids in differential diagnosis, prediction of outcome or response to molecular-targeted therapies. These are prime examples for translational cancer research. The last decade has witnessed some significant improvements in the use of this technology. This review provides an overview on the current status of IHC as applied in translational cancer research, commenting on the underlying principles in specimen preparation, reagent choice, staining procedure, and results evaluation so that both beginners and seasoned users could appreciate the key factors and benefit from this update.

Keywords: Antigen retrieval, computer-assisted image analysis, human protein atlas, immunohistochemistry, polymer-based method

How to cite this article:
Shi Z, Stack M S. An Update on Immunohistochemistry in Translational Cancer Research. Cancer Transl Med 2015;1:115-22

How to cite this URL:
Shi Z, Stack M S. An Update on Immunohistochemistry in Translational Cancer Research. Cancer Transl Med [serial online] 2015 [cited 2020 Aug 6];1:115-22. Available from: http://www.cancertm.com/text.asp?2015/1/4/115/163802

  Introduction Top

Immunohistochemistry (IHC) refers to the technology of detecting a protein or antigen of interest with a specific antibody and visualizing it on tissue section with the aid of a chromogen and microscopy. IHC can be used on paraffin-embedded sections, frozen sections, or traditional cytological smears, and liquid-based cytological preparations. [1] Immunofluorescence is a specialized form of IHC in a broader sense, but is not covered in this review. The use of IHC has been a routine and indispensable facet of modern diagnostic cancer pathology practice. [2],[3] In research settings, it has been widely used in cancer research for an even longer time. [4] Newer IHC biomarkers are being continuously found as indispensable aids in differential pathologic diagnosis, prognosis or prediction of response to molecular-targeted therapies. [5],[6] For example, IHC detection of the p16 is used as a surrogate marker for human papilloma virus (HPV) infection and HPV-related cancer. [7] BRAF V600E mutation could be detected by IHC in tumor tissues as an indicator for treatment with BRAF inhibitors. [8] These are prime examples for the success of translational cancer research. Many of us are eager to facilitate the transition of new knowledge from basic research into clinical use with this approach.

In theory, IHC is very simple. A typical workflow of IHC for research users often starts with tissue sections, followed by antigen retrieval and background suppression, application of primary and secondary antibodies, chromogenic development, and culminates with image capture, and analysis [Figure 1]. General protocols can be found readily online. [9] To some research users, IHC analysis is less favored as we increasingly embrace newer technologies such as next-generation sequencing, super-resolution microscopy, proteomic mass spectrometry, and others in our daily research activities. However, the analytical power of the unique combination of molecular detection within the context of tissue morphology that IHC brings us cannot be underestimated. Plenty of valuable information is hidden in tissue samples and may contribute tremendously to personalized or precision medicine. Meanwhile, it is not uncommon in research labs that misunderstanding of the principles of IHC leads to failure of assays or misinterpretation of IHC results.
Figure 1: Flow chart of an IHC procedure on the formalin-fixed paraffin-embedded section. Starting from tissue fixation in 10% buffered formalin, immunohistochemistry shares many of the standard steps in classic histotechnology. Antigen retrieval (heat-induced epitope retrieval), choice of the primary antibody, and polymer-based detection system (secondary antibody) are critical for the success of IHC. After antibody incubations, plenty washing steps with phosphate buffered saline should follow but use Tris-buffered saline instead when working with alkaline phosphatase-conjugated antibodies. Digital pathology solutions have made image analysis quantitative in IHC. IHC: Immunohistochemistry

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Occasional users of IHC may have not kept abreast with the recent advances in IHC technology. For cancer investigators who do not specialize in this approach, an overview of the current status of IHC as it is used in research would be beneficial since quite some recent developments in this field have been made. It should be noted that manual IHC staining is still common in most research labs, but automatic IHC devices have been widely adopted in high-volume clinical labs and core facilities, which could be integrated with other automated systems in histologic workflow for better efficiency. [10],[11] Dako and Ventana are the two major providers for automatic IHC stainers. Standardization of procedures, labor savings, better intra-lab and batch-to-batch assay consistency, and less human errors during the procedure are the benefits that come with automation. [12] Regardless of whether the procedures are manual or automatic, the principles for performing high-quality IHC are the same.

  Proper Tissue Handling and Section Preparation Top

Preanalytic factors such as delayed time to fixation and poor tissue handling impose a major negative impact on any downstream assays including IHC. [13] Inappropriate tissue preparation will make IHC inconsistent, difficult or impossible to perform. [14] Certain treatment like decalcification of bony tissues may damage your antigen of interest. No single fixative suits all needs, but 4% neutral buffered formaldehyde (aka 10% formalin) is the fixative of choice for the most applications. For those who make use of archived materials from clinical pathology departments, this is the default fixative in which your specimen was preserved. For the majority of tissue types, formalin-fixed paraffin-embedded (FFPE) tissue sections provide the standard morphological context for most applications. Although fixation, processing and embedding are usually a standardized process within a lab, when specimens from multiple centers are retrospectively collected and evaluated, quality issues may arise. Long-term room temperature storage may negatively affect IHC staining too. Thus, retrospective studies rely on existing quality control systems at source institutions. In recent years, because of the increasing demand for better RNA integrity in tissue specimens, new types of fixatives, such as PAXgene, Allprotect, and RNAlater were introduced to the research community. [15] Results suggest that PAXgene is superior in the simultaneous preservation of morphology, protein and nucleic acid molecules, but a systematic replacement of formaldehyde fixation in the clinical setting may not happen soon. Such a switch from formalin to PAXgene fixation would require a re-evaluation of IHC markers and staining procedures originally developed for FFPE tissues. [15],[16],[17]

Cross-linking between proteins caused by formalin fixation was a major problem, as this cross-linking blocks the access of the antibody to epitopes, so the frozen section was often used in the early days of IHC. This problem was essentially eliminated by the use of antigen retrieval techniques on FFPE tissue sections, a revolutionary step initiated by Shi et al. [18] in 1991. Some prefer to call this procedure heat-induced epitope retrieval (HIER). Before the advent of this technique, proteolytic enzymes were used to unmask antigens on FFPE tissue sections, but with minimal success. The current commonly used antigen retrieval method is a heat treatment in citrate buffer (10 mM sodium citrate, pH 6.0). For this incubation, standard laboratory water baths, specialized HIER devices, and many kinds of kitchen grade equipment such as vegetable steamer, pressure cooker, or microwave oven have been successfully used. The key is the right length of heat treatment of sections in a conducive buffer system. The success of HIER in IHC should be transferable to mass spectrometry based detection on FFPE materials, which would greatly facilitate the discovery of cancer-related biomarkers. [19]

  Blocking Steps to Prevent Endogenous Artifacts and Nonspecific Background Top

Since the use of enzyme-linked antibody conjugate is essential to IHC, inhibition of the related endogenous enzymes present in tissue section is an important step in the process. Historically, IHC was referred to as "immunoenzyme" or "immunoperoxidase" technology, as horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the most commonly used enzymes for visualizing positive signals in IHC. [3] To avoid interference, tissue endogenous peroxidase or AP must be suppressed. AP cannot survive the standard processing procedure for FFPE materials, but on frozen section, AP is a concern. AP-rich tissues include liver, bone, intestine, placenta, white blood cells, and some tumors. [20] Various solutions, for example, 1 mM levamisole, 0.5 M HCl, or 1 M citric acid could be used to block endogenous AP. Endogenous peroxidase is more of a concern because it survives the processing procedure and is abundant on FFPE sections. Peroxidase is rich in erythrocytes, granulocytes, macrophages, hepatocytes, neurons, and many cancer cells. Effective suppressive solutions are 3% hydrogen peroxide (H 2 O 2 ) in phosphate buffered saline or 0.024 M HCl in ethanol. [21],[22]

Endogenous biotin is a prominent concern when using the avidin-biotin complex (ABC) method in performing IHC. [23],[24] Biotin is a Vitamin B, which is used in certain cell culture media and is rich in the liver, kidney, spleen, and many cancer tissues. Antigen retrieval techniques make the presence of biotin even more problematic in these tissues. [25] However, the solution to this problem is simple: use the polymer-based method instead of ABC method for detection. In the rare situation that a biotin-containing detection or signal amplifying system must be used, it is advisable to use proportionally prepared avidin-biotin solution to block endogenous biotin. A cheaper alternative is the use of dilute egg white as an avidin source. [26],[27]

Nonspecific background staining was traditionally attributed to ionic and hydrophobic interactions between tissue molecules and antibodies, and the presence of Fc receptor on the membrane of certain types of cells such as macrophages, monocytes, granulocytes, lymphocytes, and others. [28] To overcome these factors, 5% bovine serum albumin or 2-5% normal serum from the animal species from which the secondary antibody was derived has been routinely used in standard IHC procedures (commonly incubated at room temperature for 30-60 min). For Fc receptor in particular, one may use Fab or F (ab) 2 fragment instead of whole IgG to avoid binding to Fc receptors. Interestingly, a recent study by Buchwalow et al.[28] found that in the absence of a dedicated protein or serum blocking step prior to application of primary antibody, no background staining appeared in their testing of more than 40 antibodies commonly used in pathology practice. In this study, the polymer-based methods, Dako EnVison + System and SDT AmpliStain TM were used, but it is not clear what diluent was used for the primary antibody, which often is protein or serum containing solution. In other words, simultaneous blocking at the time of primary antibody incubation may be equally effective. As far as Fc receptor is concerned, they paid special attention to bone marrow, spleen, tonsil, and blood cell smear and no unwanted background was spotted. They conclude that endogenous Fc receptors on FFPE sections do not bind to IgG antibodies in IHC. These findings provide a compelling rationale to further shorten the time for IHC procedures. It seems that the improved quality of primary antibodies over the years has made the nonspecific background less of a concern nowadays. However, when such a concern does arise, preimmune serum or IgG from the species in which primary antibody was derived could be used as a control to verify or exclude the suspicion on the antibody. [29]

  Primary Antibody is The Cornerstone of Key Reagents Top

In the early history of IHC, the tracer used for visualizing antigen-antibody complex was conjugated directly to the antibody that binds to an antigen of interest, the so-called direct method. [4] This was soon replaced by the indirect method, that is, the use of a secondary antibody for detection while primary antibody is unconjugated [Figure 2]. The secondary antibody is developed in an animal species other than the animal source of the primary antibody and recognizes the Fcg portion of a primary antibody. It should be noted that IHC detection is not limited to protein or protein in its wild-type form. Nonprotein antigen, mutant protein or other forms of modification like phosphorylation could be detected as long as a specific antibody is available. No any other reagent is more important than the right primary antibody to be chosen in IHC. No optimization of any other steps can make up for a faulty primary antibody. This is the most key reagent in IHC. While there are usually many commercially available antibodies on the market for a particular antigen, good antibodies with high specificity and sensitivity are not always obvious and may not even exist for your protein of interest. Most vendors label the antibody with "IHC-P" to indicate that this antibody could be used for IHC on FFPE materials, but that is, not indicative of a high-titer antibody with good sensitivity and specificity.
Figure 2: Scheme of polymer-based two-step method in immunohistochemistry. After antigen retrieval and proper blocking steps, the tissue section is first incubated with primary (1st) antibody. The polymeric conjugated secondary antibody (2nd) is applied afterward. In the EnVision complex, the dextran polymer bears up to 100 molecules of the enzyme (horseradish peroxidase or alkaline phosphatase) and up to 20 molecules of secondary antibody. This detection system is much more sensitive than older methods (peroxidase anti-peroxidase or avidin-biotin complex methods). Since no avidin-biotin is involved, clearer background is achieved without the need of steps to block endogenous biotin. HRP: horseradish peroxidase; AP: alkaline phosphatase

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When choosing a primary antibody for IHC, the specificity is of top priority. Unlike in western blot assay wherein nonspecific positive staining can be evaluated in the context of electrophoretic mobility of the protein of interest, nonspecific binding is difficult to define in the tissue context. Mouse monoclonal antibodies are preferred by some users because of consistent quality control from batch to batch in terms of specificity; however, rabbit polyclonal antibodies are long known to possess higher affinity. In recent years, monoclonal antibodies from rabbit have become available and increasingly popular among IHC users because of their unique high affinity, high sensitivity and high specificity. The superiority of rabbit antibodies has to do with the unique rabbit immune system, which responds to a broader range of antigens, and has more somatic gene conversion, longer, and more heterogeneous CDR3 sequences. [30] The high quality of rabbit monoclonal antibodies is indeed impressive as we experienced. Compared to mouse monoclonal antibodies to the same antigen, rabbit monoclonal provide cleaner staining, with lower or no background. [31]

Some have proposed in a recent comment in Nature to standardize the production of antibodies used in research due to significant concerns over the reproducibility issues. [32] It is troubling to contemplate that among those commonly used and commercially marketed antibodies, less than half of them recognized only their specified targets. [33] Recognizing only the intended antigen is definitely the minimum requirement for antibodies used in IHC. Polyclonal antibodies were shown to vary widely in quality. Even the long-term reliability of monoclonal antibodies is in doubt. The authors, including 110 co-signatories, call for an international initiative to use recombinant antibodies with defined binding sequences from proven hybridoma cell lines to ensure ultimate quality control. [32]

Another seemingly trivial but often neglected and serious issue related to the choice of antibody is the use of mouse monoclonal antibody on mouse tissue for IHC in the commonly used indirect scheme [Figure 2], which produces heavy background staining (note that using a rabbit primary on rabbit tissue would have the same problem). [29] This is because the secondary antibody is anti-mouse IgG, which would recognize all the endogenous IgG within mouse tissues, such as interstitial fluid, plasma, B cells, and macrophages. This problem may puzzle a user who previously only stained human tissues. The simplest way to avoid this problem is to use antibodies from another species, for example, rabbit polyclonal or monoclonal antibody on mouse tissues. When only a mouse antibody is available, the unconjugated monovalent Fab fragment from anti-mouse IgG can be used to block the binding sites on endogenous mouse IgG before applying the mouse primary antibody. Ready-to-use kits for such solutions are commercially available, for example, the M.O.M. kit from Vector Labs. Alternative solutions include the use of haptenylated primary antibodies, like those with biotin or digoxigenin, followed by secondary antibodies recognizing the corresponding hapten. [26]

  Choosing the Immunohistochemistry Markers Top

A lengthy list of proteins is being routinely used as great IHC markers in diagnostic pathology for various needs, such as differential diagnosis of cancer with unknown origin, molecular classification, prognosis or predicting therapeutic response. [5],[34],[35],[36] This is a complex topic not to be covered in detail here due to space limitation, although it is of relevance to translational cancer research. This list of markers keeps growing and is being updated constantly; those interested may refer to some useful reference books. [2],[3] For IHC-centered translational studies, researchers' interest lies in the characterization of proteins that are yet to be systematically investigated in human cancer tissues. Many of these antigens provide interesting and valuable research topics and may end up with proven clinical utility.

Worthy of mention to cancer researchers is that there are some well-established IHC markers for measuring the growth potential of a tumor or the percentage of cell death or apoptosis therein. [37],[38] The nuclear protein Ki67 has been shown to be exclusively expressed in proliferating cells and is an excellent IHC marker for tumor cell proliferating status. In many types of cancer, the Ki67 index has been routinely used as a parameter for prognosis. [37] It is also in use for certain differential diagnosis and diagnosis. PCNA (proliferating cell nuclear antigen, auxiliary protein of DNA polymerase delta) is often used for this purpose as well. In contrast to cell proliferation, the percentage of cell death or apoptosis in cancer tissue is also important to observe. The most classical measurement for apoptotic cells on tissue section is TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling), detecting DNA fragmentation by labeling the terminal end of nucleic acids cleaved by caspase-dependent mechanisms. TUNEL uses IHC components but is not a pure IHC procedure. Some frequently used apoptosis markers in IHC include cleaved cytokeratin-18 (Roche brand name is M30), cleaved caspase 3, cleaved lamin A, phosphorylated histone H2AX, and the cleaved poly (ADP-ribose) polymerase. [38] In a sense, measuring tumor growth potential and apoptotic index with IHC approaches should be as standard as measurement of tumor size/volume.

Due to recent clinical successes with immune checkpoint inhibitor therapies, immune-profiling of cancer tissues is gaining new attention. [39] Galon et al.[40] together with a worldwide consortium proposed the concept of "immunoscore" as a component of the cancer classification system. IHC staining of CD3 (pan-T cell marker) and CD8 (cytoxic T cell marker) coupled with digital image analysis of in-tumor region and invasive margin provides a scoring system in colorectal carcinoma to better stratify patients for therapeutic benefits. As many of the lymphocyte IHC markers have been well validated and used in the study and clinical diagnosis of lymphomas for many years, it can be predicted that a transition into use of these reagents for profiling cancer tissues in general will not be technically challenging.

One great resource when considering which IHC marker to study is the Human Protein Atlas project, which was inspired to provide a full coverage of each protein in the human proteome with IHC method on the all major human tissues and organs, and all major types of cancer tissues as well. [41],[42],[43] The current release (13 th version) contains protein data for 83% of all the predicted human genes. That is corresponding to 16,975 protein-encoding genes analyzed based on 24,028 antibodies, in the format of more than 13 million images from IHC revealing their expression and localization in normal tissue, cancer, cell lines, and subcellular compartment. [41],[42],[43],[44] Indeed, many members of the human proteome have not yet been characterized with a dedicated IHC study. With the expectation that every protein will be inspected by IHC in human normal and cancer tissues in this Human Protein Atlas project and appear in the database, it is advisable to consult this resource prior to each new IHC study, particularly when little or no additional published data are available. The ultimate value of reference of each stain heavily relies on the primary antibodies used.

  Detection with Polymer-Based Two-Step Method and Signal Amplification Top

To visualize the presence and abundance of a specific antigen-antibody complex, a detection system is needed. The polymer-based two-step method is currently the procedure of choice. In this scheme, as illustrated in [Figure 2], secondary antibody directed against IgG from the source animal species for primary antibody (for example, goat anti-mouse IgG) and numerous HRP molecules are conjugated on a backbone of dextran polymer. The signal from one molecule of primary antibody could thus be amplified up to 100-fold in this system. EnVision from Dako Corp., ImmPRESS from Vector Labs and OptiView from Ventana Corp., are representative products using this method. Previous methods, such as ABC method, peroxidase anti-peroxidase method or their numerous variants are now outdated due to relative lack of sensitivity, more complex protocols, and the need to block endogenous biotin in many types of tissues.

Tyramide signal amplification (TSA) is a powerful system that may be incorporated into any IHC schemes using HRP. [45],[46] It is also called catalyzed reporter deposition technique. This system takes advantage of the reaction in which the phenol moiety of labeled tyramide gets oxidized in the presence of H 2 O 2 and HRP (conjugated to secondary antibody), and produces a quinone-like structure with a radical on the C2 group, becoming "activated." Activated tyramide then rapidly and covalently binds to all nearby electron-rich residues within proximity to the initially immobilized antigen-antibody complex. [22] Such a reaction is quickly done within 10 min and amplifies the antigenic site up to 100-fold with no loss in resolution. Tyramide has desirable properties in that it is an excellent substrate of HRP since it could be conjugated to biotin, fluorescein, and many other fluorophores. In combination with the avidin-biotin system or anti-fluorescein antibody, or the use of fluorescence microscope, TSA is very versatile, and powerful in amplifying weak signals in IHC. [45] A caveat with this system is that a slight amount residual of endogenous peroxidase, not obvious in other systems, would also be amplified to high levels using this approach, and such a complete suppression of endogenous peroxidase is mandatory.

Detecting multiple antigens in the same tissue section (multiplexed staining) is also possible in IHC with bright field microscopy [Figure 3]d although it is not as commonly used as in immunofluorescence. [47] Technical feasibility comes from the fact that different enzymes (either HRP or AP) can be conjugated to different secondary antibodies, and multiple different chromogenic substrates are available to these enzymes. Using a highly sensitive detection system with no background, as the polymer-based two-step method would do, is essential to a successful multiplexed staining. Peroxidase substrates, such as DAB, DAB/Ni, AEC, NovaRED, and VIP produce sharper, denser and tightly localized precipitates. AP substrates tend to be more diffuse and translucent than peroxidase substrates. In a sequential multiplexed staining scheme, which is preferred, proper color development of the first substrate usually prevents the subsequent antibodies and detection reagents from reacting with antibody and components used to stain the previous antigen. This feature therefore makes it possible that even antibodies raised in the same species could be used for multiplexed staining in IHC. The limitation is also obvious, as unlike in immunofluorescence, overlapping colors should not be used as they cannot be differentiated using bright field microscopy. Thus, antigens to be detected with multiplexed IHC should ideally be located in different cell types, or at least in different cellular compartments (such as cellular membrane vs. nucleus) when they are in the same type of cell. The proper choice of substrates is important for a good contrast.
Figure 3: Positive staining patterns in immunohistochemistry. (a) Membranous staining: HER2 on invasive breast carcinoma, (b) nuclear staining: PCNA on mouse ID8 ovarian cancer, (c) cytoplasmic staining: CD68 on the human tonsillar tissue. In (a), (b) and (c), DAB was used as horseradish peroxidase substrate, hematoxylin as a counterstain, but images were taken from different and unrelated immunohistochemical studies. (d) Multiplex staining with CD3 as blue (VECTOR® SG), PD-L1 as brown (DAB) and nuclei counterstained with methyl green on oropharyngeal squamous cell carcinoma. Scale bar = 100 μm

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  Design, Interpretation and Quantification of Immunohistochemistry Stains Top

The procedure of performing IHC staining may seem disarmingly simple, but as in all scientific efforts, proper design is critical for a successful IHC study. For human cancer specimen-based studies, sample size, and ethics-related regulatory affairs must be properly considered. Validation of new antibodies for their first use in IHC is beyond the scope of this report. From a technical perspective, starting with a primary antibody that has been well validated for IHC application on FFPE materials is important. Users must still carefully work up the optimal conditions in-house with materials available to them. A series of dilutions of primary antibody and the secondary antibody should be tested before an optimal dilution factor is chosen. Positive tissue control, negative tissue control, and a negative reagent control (without the use of primary antibody) should be routinely included in any batch of IHC staining. Due to the existence of heterogeneous cell populations in tissue sections, the positive control and negative control may be the same tissue section. Multiple-tissue arrays are very useful as controls in IHC.

Evaluating the staining results of IHC and assigning a grade or score is the last step in the entire workflow. For this task, one must be familiar with the normal and pathological histological features of the tissue under investigation. For human cancer materials, the proper interpretation of IHC stains requires a qualified pathologist in conjunction with an examination of routine hematoxylin and eosin stained slide. Knowledge of the antigen localization is a must. There are too many presentations of false positive stains, such as edge and trapping artifacts, chromogen freckles, bubble artifacts, drying artifacts, hemosiderin or melanin pigmentation, and artifacts of poor fixation that should all be excluded in scoring. [24],[48] Control slides, inherent controls on your testing slides, proper localization of staining and differential staining from cell to cell are the key factors in judging the staining results. Except for those extracellular components, which appear in interstitial spaces, common patterns of cellular staining include the following [Figure 3]a-c: (1) Membranous staining: continuous or fragmented; (2) nuclear staining: diffuse or nucleolar; (3) cytoplasmic staining: diffuse, paranuclear or perinuclear. However, co-existence of cytoplasmic and nuclear or cytoplasmic and membranous staining is not uncommon. Grading IHC staining is often based on both staining intensity (1+, 2 + or 3 + corresponding to light, moderate, and strong staining) and percentage of positive stained cells. The final "score" of IHC staining is usually a combination of both intensity and percentage of positive cells and is computed with a defined algorithm, which often is not a set of universal criteria followed by all users but rather varies from study to study in researching settings. In clinical practice, however, CLIA has well-defined guidelines on IHC scoring for clinical use. [49],[50]

Computer-assisted image analysis on pathological images including IHC has been pursued for many years. [51],[52] The consistency in counting events, such as the number of positively stained cells, and in judging intensity by software is superior to human efforts in these regards. Many stand-alone generic or specialized software tools are available, commercially or free of charge. [53] The recent development in using whole slide image scanner, the so-called "digital pathology" solutions has made these tools available in a more integrated manner in most research centers. [54] Dominant brand names on the market for whole slide scanner/imaging include Leica's Aperio, Hamamtuso's NanoZoomer and PerkinElmer's Lamina. All of them provide corresponding software tools for analyzing and quantifying IHC staining results in addition to other digitalized pathology solutions. Our experience with Aperio's system confirms the value of these image analysis tools in quantifying large amounts of IHC data [Figure 4].
Figure 4. Image analysis software tools facilitate quantification of immunohistochemistry staining. (a) Original scanning image of Ki67 immunohistochemistry staining acquired with Aperio ScanScope CS on a tissue from oral squamous cell carcinoma, DAB as a substrate and counterstained by hematoxylin. (b) Markup image after analysis with a macro made from the nuclear staining algorithm provided in the package of image analysis tools. Blue: negative (0+) nuclei; Yellow: 1 + nuclei; Orange: 2 + nuclei; Red: 3 + nuclei. Cell counting of each category and the size of selected area are automatically reported too. The green line indicates the cancer area selected by a pathologist. Scale bar = 100 μm

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It should be noted that the human factor is still key to interpreting and analyzing IHC staining results. [55] No software could be trusted to automatically differentiate cancer from the noncancerous on tissue sections. In clinical settings, IHC staining is usually performed by histotechnologists while the results are interpreted by qualified pathologists. Automation and image quantification tools are intended to help, not replace, the efforts of pathologists. In the setting of translational cancer research, a collaboration between basic researchers and pathologists is essential due to the high degree of variation and complexity in the morphologic presentation of cancerous tissues.

In conclusion, the technology of IHC has never stopped evolving. Refinements in primary antibody quality, especially the increased use of rabbit monoclonal antibodies and efforts in standardization have built up a very solid foundation for its wide application in clinical diagnostic pathology and translational research. Antigen retrieval and highly sensitive signal detection and amplification have made IHC more accessible and user-friendly. Computer-assisted image analysis further increases the efficiency of this technology in research. IHC combines the advantages of histopathology and immunology, providing the unique perspective of molecular detection within morphologic context. It is a powerful tool to translational cancer researchers.

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  References Top

Wu HH, Jones KJ, Cramer HM. Immunocytochemistry performed on the cell-transferred direct smears of the fine-needle aspirates: a comparison study with the corresponding formalin-fixed paraffin-embedded tissue. Am J Clin Pathol 2013; 139 (6): 754-8.  Back to cited text no. 1
Lin F, Prichard J. Handbook of Pratical Immunohistochemistry: Frequently Asked Questions. 2 nd ed. New York: Springer; 2015.  Back to cited text no. 2
Dabbs D, Thompson L. Diagnostic Immunohistochemistry: Theranostic and Genomic Applications. 4 th ed. Philadelphia: Saunders; 2013.  Back to cited text no. 3
Coons AH, Kaplan MH. Localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody. J Exp Med 1950; 91 (1): 1-13.  Back to cited text no. 4
Lin G, Doyle LA. An update on the application of newly described immunohistochemical markers in soft tissue pathology. Arch Pathol Lab Med 2015; 139 (1): 106-21.  Back to cited text no. 5
Patel SP, Kurzrock R. PD-L1 expression as a predictive biomarker in cancer immunotherapy. Mol Cancer Ther 2015; 14 (4): 847-56.  Back to cited text no. 6
Chan JK, Ip YT, Cheuk W. The utility of immunohistochemistry for providing genetic information on tumors. Int J Surg Pathol 2013; 21 (5): 455-75.  Back to cited text no. 7
Qiu T, Lu H, Guo L, Huang W, Ling Y, Shan L, Li W, Ying J, Lv N. Detection of BRAF mutation in Chinese tumor patients using a highly sensitive antibody immunohistochemistry assay. Sci Rep 2015; 5: 9211.  Back to cited text no. 8
IHC World. Woodstock (MD): IHC World, LLC. Available from: http://www.ihcworld.com/general_IHC.htm. [Last cited on 2015 Jul 01].  Back to cited text no. 9
Prichard J, Bitting A, Myers J. Overview of automated immunohistochemistry. In: Lin F, Pritchard J, editors. Handbook of Pratical Immunohistochemistry: Frequently Asked Questions. 2 nd ed. New York: Springer; 2015. p. 31-5.  Back to cited text no. 10
Prichard JW. Overview of automated immunohistochemistry. Arch Pathol Lab Med 2014; 138 (12): 1578-82.  Back to cited text no. 11
Lin F, Chen Z. Standardization of diagnostic immunohistochemistry: literature review and geisinger experience. Arch Pathol Lab Med 2014; 138 (12): 1564-77.  Back to cited text no. 12
Hammond ME, Hayes DF, Dowsett M, Allred DC, Hagerty KL, Badve S, Fitzgibbons PL, Francis G, Goldstein NS, Hayes M, Hicks DG, Lester S, Love R, Mangu PB, McShane L, Miller K, Osborne CK, Paik S, Perlmutter J, Rhodes A, Sasano H, Schwartz JN, Sweep FC, Taube S, Torlakovic EE, Valenstein P, Viale G, Visscher D, Wheeler T, Williams RB, Wittliff JL, Wolff AC; American Society of Clinical Oncology; College of American Pathologists. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer (unabridged version). Arch Pathol Lab Med 2010; 134 (7): e48-72.  Back to cited text no. 13
Kalkman S, Barentsz MW, van Diest PJ. The effects of under 6 hours of formalin fixation on hormone receptor and HER2 expression in invasive breast cancer: a systematic review. Am J Clin Pathol 2014; 142 (1): 16-22.  Back to cited text no. 14
Staff S, Kujala P, Karhu R, Rökman A, Ilvesaro J, Kares S, Isola J. Preservation of nucleic acids and tissue morphology in paraffin-embedded clinical samples: comparison of five molecular fixatives. J Clin Pathol 2013; 66 (9): 807-10.  Back to cited text no. 15
Belloni B, Lambertini C, Nuciforo P, Phillips J, Bruening E, Wong S, Dummer R.Will PAXgene substitute formalin? A morphological and molecular comparative study using a new fixative system. J Clin Pathol 2013; 66 (2): 124-35.  Back to cited text no. 16
Gündisch S, Slotta-Huspenina J, Verderio P, Ciniselli CM, Pizzamiglio S, Schott C, Drecoll E, Viertler C, Zatloukal K, Kap M, Riegman P, Esposito I, Specht K, Babaryka G, Asslaber M, Bodó K, den Bakker M, den Hollander J, Fend F, Neumann J, Reu S, Perren A, Langer R, Lugli A, Becker I, Richter T, Kayser G, May AM, Carneiro F, Lopes JM, Sobin L, Höfler H, Becker KF. Evaluation of colon cancer histomorphology: a comparison between formalin and PAXgene tissue fixation by an international ring trial. Virchows Arch 2014; 465 (5): 509-19.  Back to cited text no. 17
Shi SR, Key ME, Kalra KL. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 1991; 39 (6): 741-8.  Back to cited text no. 18
Fowler CB, O′Leary TJ, Mason JT. Toward improving the proteomic analysis of formalin-fixed, paraffin-embedded tissue. Expert Rev Proteomics 2013; 10 (4): 389-400.  Back to cited text no. 19
Tesch GH, Lan HY, Nikolic-Paterson DJ. Treatment of tissue sections for in situ hybridization. Methods Mol Biol 2006; 326: 1-7.  Back to cited text no. 20
Radulescu RT, Boenisch T. Blocking endogenous peroxidases: a cautionary note for immunohistochemistry. J Cell Mol Med 2007; 11 (6): 1419.  Back to cited text no. 21
Shi Z, Johnson JJ, Stack MS. Fluorescence in situ hybridization for MicroRNA detection in archived oral cancer tissues. J Oncol 2012; 2012: 903581.  Back to cited text no. 22
Bussolati G, Leonardo E. Technical pitfalls potentially affecting diagnoses in immunohistochemistry. J Clin Pathol 2008; 61 (11): 1184-92.  Back to cited text no. 23
Bussolati G, Gugliotta P, Volante M, Pace M, Papotti M. Retrieved endogenous biotin: a novel marker and a potential pitfall in diagnostic immunohistochemistry. Histopathology 1997; 31 (5): 400-7.  Back to cited text no. 24
Kim SH, Jung KC, Shin YK, Lee KM, Park YS, Choi YL, Oh KI, Kim MK, Chung DH, Son HG, Park SH. The enhanced reactivity of endogenous biotin-like molecules by antigen retrieval procedures and signal amplification with tyramine. Histochem J 2002; 34 (3-4): 97-103.  Back to cited text no. 25
Buchwalow IB, Bocker W. Immunohistochemistry: Basics and Methods. Heidelberg: Springer; 2010.  Back to cited text no. 26
Miller R, Kubier P. Blocking of endogenous avidin-binding activity in immunohistochemistry-the use of egg white. Appl Immunohistochem 1997; 5: 63-6.  Back to cited text no. 27
Buchwalow I, Samoilova V, Boecker W, Tiemann M. Non-specific binding of antibodies in immunohistochemistry: fallacies and facts. Sci Rep 2011; 1: 28.  Back to cited text no. 28
Ward JM, Rehg JE. Rodent immunohistochemistry: pitfalls and troubleshooting. Vet Pathol 2014; 51 (1): 88-101.  Back to cited text no. 29
Kingsley M. Rabbit Monoclonal Antibody: a New Diagnostics Technology. IVD technology. Available from: http://www.mddionline.com/print/10742. [Last cited 2013 Jun 01].  Back to cited text no. 30
Rossi S, Laurino L, Furlanetto A, Chinellato S, Orvieto E, Canal F, Facchetti F, Dei Tos AP. Rabbit monoclonal antibodies: a comparative study between a novel category of immunoreagents and the corresponding mouse monoclonal antibodies. Am J Clin Pathol 2005; 124 (2): 295-302.  Back to cited text no. 31
Bradbury A, Pluckthun A. Reproducibility: standardize antibodies used in research. Nature 2015; 518 (7537): 27-9.  Back to cited text no. 32
Berglund L, Bjorling E, Jonasson K, Rockberg J, Fagerberg L, Al-Khalili Szigyarto C, Sivertsson A, Uhlén M. A whole-genome bioinformatics approach to selection of antigens for systematic antibody generation. Proteomics 2008; 8 (14): 2832-9.  Back to cited text no. 33
Chen ZE, Lin F. Application of immunohistochemistry in gastrointestinal and liver neoplasms: new markers and evolving practice. Arch Pathol Lab Med 2015; 139 (1): 14-23.  Back to cited text no. 34
Liu H. Application of immunohistochemistry in breast pathology: a review and update. Arch Pathol Lab Med 2014; 138 (12): 1629-42.  Back to cited text no. 35
Lin F, Liu H. Immunohistochemistry in undifferentiated neoplasm/tumor of uncertain origin. Arch Pathol Lab Med 2014; 138 (12): 1583-610.  Back to cited text no. 36
Pathmanathan N, Balleine RL. Ki67 and proliferation in breast cancer. J Clin Pathol 2013; 66 (6): 512-6.  Back to cited text no. 37
Holubec H, Payne CM, Bernstein H, Dvorakova K, Bernstein C, Waltmire CN, Warneke JA, Garewal H. Assessment of apoptosis by immunohistochemical markers compared to cellular morphology in ex vivo-stressed colonic mucosa. J Histochem Cytochem 2005; 53 (2): 229-35.  Back to cited text no. 38
Massi D, Brusa D, Merelli B, Ciano M, Audrito V, Serra S, Buonincontri R, Baroni G, Nassini R, Minocci D, Cattaneo L, Tamborini E, Carobbio A, Rulli E, Deaglio S, Mandalà M. PD-L1 marks a subset of melanomas with a shorter overall survival and distinct genetic and morphological characteristics. Ann Oncol 2014; 25 (12): 2433-42.  Back to cited text no. 39
Galon J, Mlecnik B, Bindea G, Angell HK, Berger A, Lagorce C, Lugli A, Zlobec I, Hartmann A, Bifulco C, Nagtegaal ID, Palmqvist R, Masucci GV, Botti G, Tatangelo F, Delrio P, Maio M, Laghi L, Grizzi F, Asslaber M, D′Arrigo C, Vidal-Vanaclocha F, Zavadova E, Chouchane L, Ohashi PS, Hafezi-Bakhtiari S, Wouters BG, Roehrl M, Nguyen L, Kawakami Y, Hazama S, Okuno K, Ogino S, Gibbs P, Waring P, Sato N, Torigoe T, Itoh K, Patel PS, Shukla SN, Wang Y, Kopetz S, Sinicrope FA, Scripcariu V, Ascierto PA, Marincola FM, Fox BA, Pagès F. Towards the introduction of the ′Immunoscore′ in the classification of malignant tumours. J Pathol 2014; 232 (2): 199-209.  Back to cited text no. 40
Uhlén M, Fagerberg L, Hallström BM, Lindskog C, Oksvold P, Mardinoglu A, Sivertsson Å, Kampf C, Sjöstedt E, Asplund A, Olsson I, Edlund K, Lundberg E, Navani S, Szigyarto CA, Odeberg J, Djureinovic D, Takanen JO, Hober S, Alm T, Edqvist PH, Berling H, Tegel H, Mulder J, Rockberg J, Nilsson P, Schwenk JM, Hamsten M, von Feilitzen K, Forsberg M, Persson L, Johansson F, Zwahlen M, von Heijne G, Nielsen J, Pontén F. Proteomics. Tissue-based map of the human proteome. Science 2015; 347 (6220): 1260419.  Back to cited text no. 41
Ponten F, Jirstrom K, Uhlen M. The Human Protein Atlas - A tool for pathology. J Pathol 2008; 216 (4): 387-93.  Back to cited text no. 42
Uhlén M, Björling E, Agaton C, Szigyarto CA, Amini B, Andersen E, Andersson AC, Angelidou P, Asplund A, Asplund C, Berglund L, Bergström K, Brumer H, Cerjan D, Ekström M, Elobeid A, Eriksson C, Fagerberg L, Falk R, Fall J, Forsberg M, Björklund MG, Gumbel K, Halimi A, Hallin I, Hamsten C, Hansson M, Hedhammar M, Hercules G, Kampf C, Larsson K, Lindskog M, Lodewyckx W, Lund J, Lundeberg J, Magnusson K, Malm E, Nilsson P, Odling J, Oksvold P, Olsson I, Oster E, Ottosson J, Paavilainen L, Persson A, Rimini R, Rockberg J, Runeson M, Sivertsson A, Sköllermo A, Steen J, Stenvall M, Sterky F, Strömberg S, Sundberg M, Tegel H, Tourle S, Wahlund E, Waldén A, Wan J, Wernérus H, Westberg J, Wester K, Wrethagen U, Xu LL, Hober S, Pontén F. A human protein atlas for normal and cancer tissues based on antibody proteomics. Mol Cell Proteomics 2005; 4 (12): 1920-32.  Back to cited text no. 43
Uhlen M, Oksvold P, Fagerberg L, Lundberg E, Jonasson K, Forsberg M, Zwahlen M, Kampf C, Wester K, Hober S, Wernerus H, Björling L, Ponten F. Towards a knowledge-based Human Protein Atlas. Nat Biotechnol 2010; 28 (12): 1248-50.  Back to cited text no. 44
Stack EC, Wang C, Roman KA, Hoyt CC. Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of tyramide signal amplification, multispectral imaging and multiplex analysis. Methods 2014; 70 (1): 46-58.  Back to cited text no. 45
McLachlan CS, Jusuf PR, Rummery N, Kummerfeld SK, Hambly B, McGuire MA, Turner V. Tyramide signal amplification enhances the detectable distribution of connexin-43 positive gap junctions across the ventricular wall of the rabbit heart. Arch Histol Cytol 2003; 66 (4): 359-65.  Back to cited text no. 46
Huang W, Hennrick K, Drew S. A colorful future of quantitative pathology: validation of Vectra technology using chromogenic multiplexed immunohistochemistry and prostate tissue microarrays. Hum Pathol 2013; 44 (1): 29-38.  Back to cited text no. 47
Ilyas M, Grabsch H, Ellis IO, Womack C, Brown R, Berney D, Fennell D, Salto-Tellez M, Jenkins M, Landberg G, Byers R, Treanor D, Harrison D, Green AR, Ball G, Hamilton P; National Cancer Research Institute (UK) Biomarker and Imaging Clinical Studies Group. Guidelines and considerations for conducting experiments using tissue microarrays. Histopathology 2013; 62 (6): 827-39.  Back to cited text no. 48
Hewitt SM, Robinowitz M, Bogen SA, Gown AM, Kalra KL, Otis CN, Spaulding B, Taylor CR. Quality Assurance for Design Control and Implementation of Immunohistochemistry Assays: Approved Guideline. 2 nd ed. Wayne: Clinical and Laboratory Standards Institute; 2011.  Back to cited text no. 49
Ramos-Vara JA, Miller MA. When tissue antigens and antibodies get along: revisiting the technical aspects of immunohistochemistry - The red, brown, and blue technique. Vet Pathol 2014; 51 (1): 42-87.  Back to cited text no. 50
Kohlberger PD, Obermair A, Sliutz G, Heinzl H, Koelbl H, Breitenecker G, Gitsch G, Kainz C. Quantitative immunohistochemistry of factor VIII-related antigen in breast carcinoma: a comparison of computer-assisted image analysis with established counting methods. Am J Clin Pathol 1996; 105 (6): 705-10.  Back to cited text no. 51
Di Cataldo S, Ficarra E, Macii E. Computer-aided techniques for chromogenic immunohistochemistry: status and directions. Comput Biol Med 2012; 42 (10): 1012-25.  Back to cited text no. 52
Prasad K, Prabhu GK. Image analysis tools for evaluation of microscopic views of immunohistochemically stained specimen in medical research - A review. J Med Syst 2012; 36 (4): 2621-31.  Back to cited text no. 53
Pantanowitz L, Valenstein PN, Evans AJ, Kaplan KJ, Pfeifer JD, Wilbur DC, Collins LC, Colgan TJ. Review of the current state of whole slide imaging in pathology. J Pathol Inform 2011; 2: 36.  Back to cited text no. 54
Berho M, Bejarano PA. Judging pathological assessment in cancer specimens. J Surg Oncol 2014; 110 (5): 543-50.  Back to cited text no. 55


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