Principles of Diagnostic Immunohistochemistry - HML 3023

Immunoenzyme Techniques

• Attaching an enzyme to a specific antibody allows enzymatic activity to induce a visible color change in the substrate and chromogen.
• This identifies the sites of localization of the antigen-enzyme complex within the tissue, indicating the site of the specific antibody-antigen reaction.

Method

• Horseradish peroxidase was the original immunoenzyme method.
• Other enzyme systems like glucose oxidase and alkaline phosphatase have also been successfully used.
• There are many commercial sources of enzyme-conjugated antibodies and chromogens, as well as kits for common multi-layered immunoenzyme staining procedures.
• Methods include:
  • Direct Method
  • Indirect Method
  • PAP Method

Direct Immunohistochemistry Technique

• $FITC$ = Fluorescein isothiocyanate
• $FITC$ reacts with a primary amine on the protein to form a covalent amide bond.
• $FITC$-labelled protein substrates/peptides, antibodies, and peptide hormones are used as specific probes in enzyme kinetics, immunocytochemistry, flow cytometry, and identification of receptors on target cells.

Direct Method

• Direct IF uses a single antibody directed against the target of interest.
• The primary antibody is directly conjugated to a fluorophore.
• The primary antibody with specificity for the antigen has the enzyme label attached directly to it through its $Fc$ portion.
• If the enzyme is peroxidase, incubation with hydrogen peroxide and a chromogen such as diaminobenzidine results in a brown reaction product localized to the site of the antigen.

Indirect Antibody Technique

• Indirect IF uses two antibodies.
• The primary antibody is unconjugated, and a fluorophore-conjugated secondary antibody directed against the primary antibody is used for detection.

Two-Step Indirect Method

• Involves a primary and secondary antibody

Indirect Method

• The primary antibody, which is unlabeled, is detected by a secondary antibody which has the bound enzyme label.
• The secondary antibody has specificity against immunoglobulin from the species which was the source of the primary antibody.

Advantages of Indirect Method

• It is not necessary to conjugate every antibody making it cheaper.
• Although there can be only one primary antibody bound to the antigen site, there can be several secondary antibodies bound to the primary antibody.
• Each of the secondary antibodies will have a conjugate, increasing the intensity so that smaller amounts of antigen can be detected.
• It is more sensitive since there can be more than one conjugated antibody bound to a single antigen site.
• The increased sensitivity means that the antibodies can be used in a more dilute solution, reducing the cost of preparing a large number of slides.

Immunoenzyme Techniques

• The problems with fluorescein labels led to a search for other antibody labels including enzymes.
• The use of these histochemical labels is termed immunohistochemistry.
• It uses enzymes to label the antibodies to identify the sites of binding
• The most popular are horse-radish peroxidase $(HRP)$ and alkaline phosphatase $(AP)$
• Since both enzyme and the antibody are proteins, they can act as antigens
• It was realized that it is possible to link these components together by using a third antibody
• This requires two different species of animal and three antibodies

The three antibodies are:

• The primary against the antigen prepared in a stated species, e.g. rabbit anti-actin
• An anti-horse-radish peroxidase prepared in the same species, e.g. rabbit anti-HRP
• A bridging antibody against the immunoglobulin of the species used to prepare 1 and 2, e.g. goat anti-rabbit Ig

Unlabelled antibody Enzyme Method

• Overview of the components and their interactions

PAP Method

• (Peroxidase anti-peroxidase method)

PAP - Indirect Method

*Indirect Method - PAP

• Figure 1. Peroxidase Anti-Peroxidase (PAP) Complex Method.

Peroxidase-anti-peroxidase technique

• This is a three-stage procedure.
• The primary antibody has a specificity for the tissue antigen.
• The secondary antibody forms a bridge between the primary antibody and peroxidase-antiperoxidase complex.
• The PAP complex comprises a soluble and stable preformed mixture of antibody against horseradish peroxidase and horseradish peroxidase antigen.
• The primary antibody and PAP complex must be from the same species so that the secondary antibody can bind specifically for the $Fc$ components of both the primary and tertiary reagents.

Controls

• Controls are needed to ensure the technique is working correctly
• A minimum of three controls are needed:
  • Known positive - to test for a protocol or procedure used. It will be ideal to use the tissue of known positive as a control.
  • Known negative - to test for the specificity of the antibody involved.
  • Control using the test section but omitting the primary antibody

Avidin-Biotin Peroxidase Method

• Standard immunohistochemistry method used widely.
• Avidin is a large glycoprotein, that can be labeled with peroxidase and has high affinity for biotin.
• Biotin is a low molecular weight vitamin that can conjugate various proteins such as antibodies
• The technique involves the use of primary unlabelled antibody which binds to the antigen of interest in the tissue of interests
• This is then recognized by a secondary, biotinylated antibody.
• The biotin on the secondary antibody is then recognized by an avidin – biotin peroxidase which is treated with a chromogenic substrate (e.g: DAB) which reacts with the peroxidase to produce various colored end products which are recognized under the microscope.

The avidin-biotin-peroxidase technique

• In this procedure the secondary antibody is biotinylated.
• Avidin has four avid binding sites for biotin and can be chemically conjugated to horseradish peroxidase.
• The tertiary complex of avidin-biotin- peroxidase molecules which localizes several molecules of peroxidase to the site of antibody- antigen reaction in the tissue section.

Avidin-biotin method

ABC Method

Indirect ABC Method

• Figure 2. Avidin-Biotin Complex (ABC) Method.

ABC method

• Steps in the ABC Method

Other considerations

*Tissue fixation and antigen preservation
*Enzyme antigen retrieval
* Microwave antigen retrieval
* Pressure-cooker retrieval
* HMAR - heat-mediated antigen retrieval

• Automation
• Counter stains
• Reagents for Immunolabelling
  • Introducing a new reagent
• Adhesives
• Controls
• Interpretation

Diagnostic applications

• Diagnostic histopathology is the art of discriminative recognition of patterns of tissue derangements in different diseases, related and unrelated.
• Rapidly become an integral part of most diagnostic histopathology service laboratories, reflecting its considerable ability to facilitate histological diagnosis
• Can be divided into two aspects:
  • A) Antigens and antibodies: their common and specific uses
  • B) Diagnostic problem solving: the areas served by these antibodies with emphasis on diagnostic algorithms

Markers for Certain Cancers

• Table of markers for various malignancies
  • Malignancy, LMW-CK, Vimentin, CD-45, HMB-45, GFAP
  • Carcinoma, +, +/-, -, -, -
  • Sarcoma, -, +, -, -, -
  • Lymphoma, -, +/-, +, -, -
  • Melanoma, -, +, -, +, -
  • Mesothelioma, +, +, -, -, -
  • Glioma, -, +, -, -, +

Diagnostic approach to tumor identification

• While immunohistochemistry is applicable for the demonstration of almost any antigenic structures in cells and tissues, its major purpose in pathology is specifically to identify lineages of cell populations in order to classify neoplastic lesions, thus serving as a supplement to the morphological criteria upon which the pathology diagnoses are based.

Diagnostic approach to tumor identification

• Immunohistochemistry has the potential of transforming tumor pathology from a subjective "art" to an objective science, making it an important tool in the everyday diagnostic work.
• The task of correct tumor classification is sometimes difficult and always critical for proper patient management.
• For example, therapies highly effective but posing serious toxicity risks are appropriate for many types of malignant lymphoma, but ineffective or even detrimental for proliferations mimicking lymphoid tumors (so- called hyperimmune reactions or atypical lymphoproliferative disorders).
• On the other hand, these therapies are insufficient for the treatment of carcinomas.

HMB45 on melanoma

• $HMB-45$ is a monoclonal antibody that reacts against an antigen present in melanocytic tumors such as melanomas, and stands for Human Melanoma Black.
• It is used in anatomic pathology as a marker for such tumors.

PSA antigen positive cells

• Elevated prostate- specific antigen $(PSA)$ levels can be a sign of prostate cancer.
• It can also indicate noncancerous problems such as prostate enlargement and inflammation.