Immunohistochemistry (IHC)

introduction:

Immunohistochemistry (IHC) employs monoclonal and polyclonal antibodies to detect, identify, and localize antigens within tissues. Tumor-specific antigens may be newly expressed or overexpressed in tissues, and their detection can provide insights into the health status of the target cells.

The IHC technique requires tissue sampling and biopsy. After processing and embedding the sample in paraffin blocks, sections are cut using a microtome and transferred onto slides. These samples are then incubated with a suitable antibody. The binding sites of antibodies to antigens can be visualized using a standard or fluorescent microscope, utilizing markers such as fluorescent dyes, enzymes, radioactive elements, or colloidal gold. These markers are directly attached to either the primary or secondary antibodies.

The fundamental concept of immunohistochemistry and its applications emerged in the 1930s, but it was not until 1941, with the publication of the first IHC study, that it became established. Coons and colleagues utilized fluorescein-conjugated antibodies to accurately locate pneumococcal antigens in infected tissues.

With the advancement of IHC techniques, enzyme labels such as peroxidase and alkaline phosphatase were introduced. Gold labeling has also been employed to identify immunohistochemical reactions at both light and electron microscopic levels. Other labels include radioactive elements, and isotopic labeling can be demonstrated through autoradiography. The goal of IHC is to achieve high-quality staining while minimizing tissue damage and using minimal antibody amounts to assess tumor levels and their associated markers.

Applications of Immunohistochemistry

Since immunohistochemistry involves specific antigen-antibody reactions, it has significant advantages over traditional staining techniques that only identify a limited number of proteins, enzymes, and tissue structures. Consequently, IHC has become an essential technique widely utilized in numerous medical research laboratories as well as in clinical diagnostics.

Here, we briefly outline nine applications of immunohistochemistry:

Prognostic Markers in Cancer

IHC is employed to determine the prognosis of tumors by identifying enzymes, tumor-specific antigens, oncogenes, tumor suppressor genes, and cellular proliferation markers. Analysis of tumors using these methods has shown considerable advancements over traditional diagnostic and clinical grading methods. IHC is also used for disease diagnosis, drug development, and biological research. By using tumor-specific markers, physicians can diagnose cancer (benign or malignant), stage and grade tumors, identify cancer cell types, and locate the source of metastasis.

Tumors of Unknown Origin

Various IHC panels facilitate the identification of tumors with an unknown origin and metastatic tumors from an unspecified primary site. The selection of antibody panels is based on clinical history, morphological characteristics, and pathology examinations on H&E-stained slides, as well as results from other relevant studies.

Predicting Treatment Response

IHC is extensively used to predict treatment responses in two distinct tumors: prostate and breast carcinomas, both of which are regulated by estrogen and androgen hormones. Specific receptors for these growth-regulating hormones are present in the corresponding tumor cells. Tumors with high levels of these receptors represent suitable drug targets for the elimination of endogenous hormones or for hormone therapy, such as estrogen therapy in prostate cancer and androgen therapy in breast cancer.

Infections

IHC methods are utilized to confirm infectious targets in infections like cytomegalovirus, hepatitis B virus, and hepatitis C virus. This identification process in tissues involves the use of specific antibodies against microbial DNA or RNA and other microbial parameters. This approach is regularly employed to validate disease targets by tracking the expression of these targets in affected tissues during disease progression.

Genetics

IHC can determine the function of specific genes in fundamental biological processes—such as apoptosis—and to assess gene expression patterns. For instance, a specific monoclonal antibody against the p53 homolog can be used to trace it in the pro-apoptotic pathway.

Neurogenic Disorders

Neurodegenerative disorders encompass a wide range of diseases characterized by dysfunction and death of vulnerable neuronal cell populations. The role of IHC is increasingly significant in classifying neurogenic disorders and developing crucial diagnostic criteria.

Traumatic Brain Injury

In recent years, immunohistochemical staining for beta-amyloid precursor protein has been validated as a method for diagnosing axonal injury within 2 to 3 hours post-injury. Immunohistochemical diagnosis of axonal injury can be used in forensic settings to establish trauma timelines.

IHC and Its Applications in Muscle Diseases

Accurate diagnosis of muscular dystrophies is critical due to the implications of hereditary diseases and their precise predictions. Recent years have confirmed multiple disruptions in muscle proteins in muscular dystrophies. Such disruptions target muscle proteins located in the sarcolemma, extracellular matrix, cytosol, nucleus, and other tissues containing muscle fibers. Skeletal muscle biopsy can play a crucial role in distinguishing vascular dystrophies from non-dystrophic disorders, and IHC can help in the specific diagnosis of different types of dystrophies based on their protein sources.

Research Applications of IHC

Current research on the causes of neurodegenerative diseases focuses on identifying factors that induce double-stranded spiral filaments, beta-amyloid deposition, cytoplasmic accumulation, and more. Thus, conducting such studies to diagnose and quantify abnormal proteins contributing to neurodegenerative diseases is of high importance. IHC, using antibodies against beta-amyloid, alpha-synuclein, ubiquitin, huntingtin, polyglutamine, and other markers, has become a common tool for precise characterization and quantitative assessment of abnormal proteins in both human and laboratory animal tissues. Consequently, IHC is a vital instrument in diagnostic and research laboratories.