Immunohistology

Author: Prof. Dr. med. Peter Altmeyer

Last updated on: 14.02.2022

Definition
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Immunohistology is a method for visualizing cell- and tissue-specific antigens (proteins) after they have been marked with specific antibodies in situ and thus visualized. The term immunohistology covers the fields of immunohistochemistry and immunostaining.

General information
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The immunohistological detection of antigens is based on the highly specific affinity of antibody binding sites to certain protein structures of the antigens (so-called antigen-antibody reaction).
Methodology: First, the histological tissue in which the protein is to be detected is processed (by means of fixation, embedding, storage and, if necessary, pre-treatment by background reduction and antigen masking). Subsequently, the primary antibodies directed against the antigen are incubated with the tissue. Ideally, specific and strong binding between antibody and epitope occurs. For visualization, the primary antibodies used are either themselves labeled with a dye or enzyme that can be visualized or are labeled via a second incubation step with a dye- or enzyme-conjugated secondary antibody that can then be visualized. Of the enzymes labeled as AK, peroxidases are particularly well suited and are widely used in routine applications. Iin electron microscopy, one uses a gold particle coupled secondary antibody,s.a. immunogold labeling.
Visual detection is achieved by fluorescence and enzymatic color reactions. The goal is to detect a signal at the site of the epitope of sufficient strength.
High specificity and affinity of the antibody is required to avoid cross-reactions with similar epitopes.
The antigen-antibody reaction depends, among other things, on temperature, concentration, incubation time and the optimal reaction milieu (pH, salt concentrations).
Direct detection of the antigen-antibody reaction: Conjugation of the protein with the antibody to which an enzyme (e.g., horseradish peroxidase) or fluorochrome (e.g., FITC, Texas-Red) is conjugated. This is followed by binding of the antibody to the antigen, and unbound antibodies are washed off. Subsequently, a substrate is offered to react with the enzyme to form a dye. This dye forms only where the immunochemical reaction has occurred. For fluorochrome-labeled antibodies, detection is done directly in the fluorescence microscope.
Indirect method: Here, a specific antibody is applied to the tissue to be examined. Subsequently, a 2nd antibody is applied, which is directed against the first antibody (so-called secondary antibody). This is coupled with an enzyme and triggers the known color formation. The reaction is thus visualized.
In the case of small amounts of protein, a tertiary antibody can be added, which couples to the secondary antibody. This step serves to amplify the signal.
The indirect technique is used, among other things, for the detection of already bound endogenous antibodies (e.g. autoantibodies, antinuclear autoantibodies, ANCA, anti-skin antibodies).

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Implementation
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General procedure for the indirect method:

Fixation of the tissue sections (e.g. in ethanol).
Incubation with hydrogen peroxide to block the activity of endogenous peroxidase.
Treatment with certain solvents to increase the permeability of the cell membranes and thus facilitate the docking of the antibodies to the epitopes.
Reduction of unspecific binding by incubation in buffer substances.
Incubation of the tissue sections (usually 60 minutes) with the first antibody.
Rinse the tissue sections after the 1st incubation
Subsequently incubate with 2. antibody for another 60 minutes.
Then detection reaction with a substrate conjugated to the peroxidase.
To visualize the reaction transfer the treated tissue into a detection solution.
Finally, transfer of the treated tissue sections from the aqueous to the organic phase by means of a staining series.
Finally observation of the staining reaction under the microscope.

Note(s)
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Commonly used enzymes and substrates:

For the detection reaction: DAB (3,3'-diaminobenzidine, forms a brown end product.
Horseradish peroxidase (HRP): marker enzyme conjugated with the antibody.

Literature
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Gambichler T et al (2007) Immunohistology of amicrobial pustulosis of the folds. Clin Exp Dermatol 32: 155-158

Tables
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Antigen	Explanations/cellular reactivity/tissue specificity
Actin	Actins are cytoskeleton proteins that are found in all eukaryotes. They form the third group of cytoskeleton proteins besides microtubules and intermediary filaments. Actin filaments are highly conserved during evolution. Together with other proteins (including myosin), actin filaments mediate movements. Actin is found in all muscle cells. In tumours derived from these tissues, actin is also produced in almost all cases. This primarily concerns leiomyomas, leiomyosarcomas and rhabdomyosarcomas. With antibodies that differentiate between smooth muscle and muscle-specific actin, corresponding soft tissue tumors can be further differentiated.
	Application: IHC (P) (G)
amyloid A	Amyloid A is an extracellularly deposited, insoluble fibrillar glycoprotein that is highly resistant to proteolytic degradation and is produced by the precursor protein serum amyloid A. Amyloidosis is the generic term for a group of diseases which share the extracellular deposition of fibrillary proteins with a specific biochemical structure known as ß-sheets.
	Application: IHC (P) (G)
bcl-2	The bcl-2 gene has been classified as a potential proto-oncogene because it is involved in the 14;18 translocation. This is also supported by proven homologies to Epstein-Barr virus gene regions. The expression of the bcl-2 protein has been demonstrated in cases of lymphomas with a 14;18 translocation. In general, positive results help in the classification of follicular lymphomas and various diffuse lymphoproliferative diseases.
	Application: IHC (P) (G)
B cell antigens	CD10, CD20, CD21, CD45RA, CD74, CDw75, CD79a, MB2, Kappa/Lambda light chains
	Application: IHC (P) (G)
CEA	Carcinoembryonic antigen is a highly glycosylated protein with a relative molecular mass of approximately 180 kDa. CEA is an important tumor marker of colorectal and other carcinomas. All members of the CEA subgroup are cell membrane-bound and show a complex expression pattern in normal and malignant tissues. Eccrine and apocrine sweat glands, Paget's disease, gastrointestinal carcinomas, microcystic adnexal carcinoma. Sebaceous glands and melanocytes (NZN and melanoma) can express CEA, occasionally Merkel cell carcinomas are positive.
	Application: IHC (P) (G)
COX-2	The cyclooxygenase enzymes COX-1 and COX-2 are of great importance in the biosynthesis of arachidonic acid prostaglandins. COX-2 is an inducible enzyme responsible for prostaglandin synthesis at the site of inflammation. COX-2 also appears to be involved in carcinogenesis processes, as specific COX-2 inhibitors have shown antitumour effects. Among other tumors, most primary malignant melanomas show expression of COX-2, while benign nevi and adjacent normal epithelium are negative.
	Application: IHC (P) (G)
Epstein-Barr virus	Epstein-Barr virus (EBV) is a human herpes virus that is usually associated with a widespread asymptomatic infection. More than 95% of the adult population worldwide are carriers of this virus. EBV-infected cells and neoplasia usually show one of three different expression patterns of the latency-associated viral genes. In type I latency, only the EBV-encoded nuclear antigen. (EBNA-1) is expressed together with two small polyadenylated nuclear ribonucleic acids (EBER 1 and 2) that occur in Burkitt's lymphoma. In type II latency, three further latent membrane proteins (LMP-1, -2A, -2B) are expressed; these are found in Hodgkin's disease and nasopharyngeal carcinoma. Type III latency occurs in lymphoproliferative diseases in immunocompromised individuals. EBV is associated with certain tumors of lymphoid and epithelial origin, including Burkitt's lymphoma, immunoblast lymphoma, T-cell lymphoma and Hodgkin's disease. The only situation where the full virus is replicated is hairy leukoplakia, which is often observed in AIDS patients.
Factor VIII	Factor VIII is a macromolecular complex consisting of a coagulation-promoting factor and the protein factor VIII-associated antigen (von Willebrand factor). The von Willebrand factor plays a central role in the blood coagulation mechanism and binds specifically to a receptor present on platelets. In pathological tissue, anti-Factor VIII-associated antigen detects the majority of malignant and benign vascular tumors.
	Application: IHC (P) (G)
gp 100	(Antibody clone HMB45 ) The antibody clone HMB45 stains a cytoplasmically localised antigen (gp100) in melanocytes and melanocytic tumours. The antigen represents a component of melanosomal oxidoreductases and is therefore melanosome-specific. HMB-45 can be detected in about 85% of malignant melanomas. Nevi and resting melanocytes are usually negative.
	Application: IHC (P) (G)
Intermediary filaments	Intermediate filaments (IF) are present in almost all types of mammalian cells. The name derives from the fact that intermediate filaments with a diameter of 10 nanometers are located between the actin filaments (7 nm) and the microtubules (25 nm). Intermediary filaments, together with the microtubules and actin filaments, form the cytoskeleton of the cell as so-called microfilaments. Five main types are distinguished: Cytokeratins are found in epithelial cells, desmines in muscle cells, neurofilaments in neurons, glial filaments in astrocytes and vimentin in mesenchymal cells. Since the distribution patterns of tissue in neoplasms are highly conserved, staining of tissue sections with antibodies of different IF types can provide useful information in the identification of different tumors.
	Application: IHC (P) (G)
Cytokeratin	Positive results support the classification of healthy and neoplastic tissues that are epithelial in origin. Cytokeratins may also be expressed in non-epithelial tumours, e.g. epithelioid angiosarcoma, leiomyosarcoma, epithelioid fibrosarcoma. Cytokeratins can be co-expressed with vimentin and other intermediary filaments, e.g. in renal cell carcinoma metastases.
	Application: IHC (P) (G)
Desmin	53 kDa heavy intermediate filament protein present in the cytoplasm of skeletal and cardiac muscle cells as well as in most smooth muscle cells. Antibodies can be used to detect cells from tumours of myogenic origin originating in smooth muscle (uterine and cutaneous leiomyomas and some gastric leiomyosarcomas) as well as in striated muscle (alveolar and embryonic rhabdomyosarcomas).
	Application: IHC (P) (G)
Neurofilament	Nerve cells (axons) of the central and peripheral nervous system form neurofilament and can be helpful in identifying tumors with neuronal differentiation as target antigen.
	Application: IHC (P) (G)
Glial fiber protein	Identification of astrocytes as well as astrocytic cells in normal and diseased conditions.
	Application: IHC (P) (G)
Vimentin	Vimentin is the cytoskeletal protein of mesenchymal cells including fibroblasts, chondrocytes, osteocytes, endothelia and white blood cells. It is generally formed in sarcomas and is used as a superior marker for soft tissue tumors. However, also de-differentiated carcinomas show expression of vimentin, then mostly with an at least partial coexpression of cytokeratins, as for example in epithelioid sarcomas.
	Application: IHC (P) (G)
Kappa light chains	Kappa light chains are amino acid polypeptide chains, each with a variable and constant domain. All five immunoglobulin classes have one of two immunoglobulin L-chains. The second light chain type is called lambda. Only one L-chain type, i.e. either lambda or kappa, is ever produced in a single cell. In the normal B cell population, 60% of the immunoglobulins belong to the type Kappa and the remaining 40% to the type Lambda. Positive results contribute to the classification of B-cell lymphomas by detecting their light chain restriction.
	Application: IHC (P) (G)
Ki 67 (MIB1)	The Ki67 antigen is a proliferation marker, i.e. it stains the cells in the tissue that are multiplying, the so-called growth fraction. During the cell cycle, this antigen is expressed in the G1, S, G2 and M phases. Resting cells, i.e. cells in the G0 phase, do not express the Ki67 antigen. The staining for Ki67 thus provides indirect information on the growth rate of a tumour and is therefore invaluable in routine diagnostics. But also in the decision between benign, preneoplastic and malignant changes the presentation of the Ki67 antigen can provide valuable assistance.
	Application: IHC (P) (G)
Collagen IV	Collagen IV is a major component of the basement membrane, especially in the walls of blood vessels, the area surrounding Schwann cells and most epithelial tissues. In congenital epidermolysis bullosa both main variants (EB simplex and EB dystrophica) can be distinguished by means of antibodies.
	Application: IHC (P) (G)
Lambda light chains	Lambda light chains are one of two amino acid residue polypeptide chains present in all five immunoglobulin classes; the other type of light chain is of the kappa type. In humans, approximately 40% of the light chains are of the lambda type and 60% are of the kappa type. Each immunoglobulin has only one type of light chain.
	Application: IHC (P) (G)
Laminin	Laminins are large basement membrane glycoproteins. There are currently 15 different isoforms known. The laminin isoforms are located at different tissue and developmental stage specific sites. Laminins promote cell adhesion, migration, protease activity, proliferation, tumor growth, angiogenesis and metastasis.
	Application: IHC (P) (G)
Mast cell tryptase	Mast cell tryptases form a family of trypsin-like, neutral serine proteases that are predominantly expressed in mast cells. All these substances are important mediators involved in bronchoconstriction and overreaction of the respiratory tract, which in turn make a significant contribution to allergic diseases of the respiratory tract. Mast cell degranulation and the subsequent release of mast cell tryptase, as well as histamines, leukotrienes and cytokines into the surrounding tissue, is a key event of an inflammatory reaction. The detection of mast cell tryptase is crucial for the identification of highly atypical, hypogranulated or even non-metachromatic mast cells, especially in mast cell leukemia, and for the detection of small mast cell infiltrates.
	Application: IHC (P) (G)
Melan-A	The gene encoding the melanoma A antigen is 18 kb long and is only expressed in normal melanocytes and in most melanoma tumors. Melan-A is a transmembrane protein of unknown function consisting of 118 amino acids.
	Application: IHC (P) (G)
Myeloperoxidase	Myeloperoxidase is a major component of the cytoplasmic primary (azurophilic) granules of neutrophil myeloid cells. The function of myeloperoxidase and other enzymes in the primary granules is to decompose microorganisms and foreign bodies within the phagocytosis vacuole of the cell. Since primary granules occur at an early stage of myeloid cell differentiation and are present throughout maturation, myeloperoxidase is a useful marker for the detection of myeloid cells.
	Application: IHC (P) (G)
p53	p53 is a tumour suppressor with a molecular weight of 53 kD. The gene product is nuclear localized and represents a central protein of cell cycle control. The central function of p53 is to prevent cells with genetic damage from multiplying. These cells then die by apoptosis. If the function of p53 is restricted or abolished by a mutation in the gene, genetically damaged cells can no longer be sorted out and genetic damage can accumulate over several cell cycles. In normal tissues, p53 is only expressed in small amounts. However, if there is a point mutation in the p53 gene that limits the function of the protein, the defective protein accumulates in the nucleus and can then be detected immunohistologically. Point mutations of the p53 gene occur very frequently in malignant tumours of different histogenesis. A strong nuclear expression in almost all tumor cells can be used as malignancy criterion.
	Application: IHC (P) (G)
S100	S100 represents a group of relatively small proteins that bind calcium ions as a common property and then activate other proteins depending on the intracellular calcium concentration. S100 proteins are found in glial and ependymal cells of the CNS. In addition, melanocytes and melanocytic tumours are S100-positive. In malignant melanomas, S100-positivity is usually still found even if other melanocytic markers are no longer formed due to progressive de-differentiation. Salivary gland tumours and tumours of the eccrine sweat glands often also show an expression of S100.
	Application: IHC (P) (G)
T cell antigens	CD3, CD4, CD5, CD8, CD43, CD45RO, CD45RA
	Application: IHC (P) (G)