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Critical evaluation of the use of indirect immunocytochemical techniques in diagnostic pathology - Essay Example

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Immunocytological techniques as applied to immunohistochemistry (IHC) is an integral technique in present-day diagnostic pathology. This technique allows one to examine cytological details and tissue architecture better. …
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Critical evaluation of the use of indirect immunocytochemical techniques in diagnostic pathology
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Download file to see previous pages Immunocytological techniques as applied to immunohistochemistry (IHC) is an integral technique in present-day diagnostic pathology. This technique allows one to examine cytological details and tissue architecture better. Antigens introduced into the tissue will bind to labeled antibodies acting as specially designed reagents, and this technique allows one to assess the presence of specific antigen or protein. This binding event is visualized and examined under a microscope. Definition, Principles, and Development of IHC: IHC is a technique of antigen localization with antibody tagging and then tracking that antibody to detect the antigen. When an antibody is labeled by a specific technique, this can act like reagents in a designed antigen-antibody reaction. This reaction can be visualized by markers, such as, fluorescent dye, enzymatic reaction, radioactive tagging, or colloidal gold. These can detect cellular antigens in tissue sections. It started with discovery of immunofluorescence technique by Coons in 1941. Since then, techniques have developed to improve the sensitivity and specificity of this diagnostic tool, which basically detects antigen-antibody binding demonstrated with coloured histochemical reactions visible by light microscopy or fluorochromes with ultraviolet light. Later, techniques were developed to use enzymes like peroxidase and alkaline phosphatase or colloidal gold and the microscopy advanced to electron microscopy to make detections better.

Direct versus Indirect Methods and Criteria for Use: Direct method is a staining

method where a labeled antibody reacts directly with the antigen in the tissue section.

This utilizes only one antibody and cannot amplify signal much. This is a rarely used

method nowadays. Indirect method, on the other hand, allows an unlabeled antibody to

react with the target antigen, and then a second antibody developed against the first

antibody is allowed to react with the primary antibody. This method has the benefit of

signal amplification through multiple secondary antibody reactions with different

antigenic sites on the primary antibody, making this method a more sensitive diagnostic

method. Direct technique can be used to diagnose Hirschprung's disease to detect

intestinal ganglion cells and nerve fibres, whereas indirect technique can be used to detect

cell proliferation in a tissue specimen.

Types of Indirect IHC Methods: Indirect immunofluorescence method, where the

antibody is labeled with a fluorescent dye, such as, FITC, Texas red, or rhodamine. The

other method available is indirect immunoenzyme method, where the antibody in the

second layer is labeled with peroxidase (PAP or peroxidase-antiperoxidase), alkaline

phosphatase (APAAP or alkaline phosphatase-anti alkaline phosphatase), glucose

oxidase, or avitin-biotin complex method (ABC).

Use of monoclonal or polyclonal antibodies: Monoclonal antibodies are raised from

clones of a single hybridoma cell and recognizes only a single epitope on the antigen;

polyclonal antibodies recognize independent epitopes on the antigen. Polyclonal

antibodies against casein component of cow's milk may be used to detect residual

antigenic activity in infant formula. Monoclonal antibodies can be used to mediate or

modulate physiological effects for research, diagnostic, or therapeutic purposes.

Tissue Preparation Prior to IHC: Fixation is important to preserve tissue

architecture. To maintain cell morphology, rapid fixation is necessary. Excessive

fixation, however, compromises antigen-binding capacity. Many antigens can be

successfully demonstrated in formalin-fixed paraffin embedded tissue sections. Cryostat

is a system of instruments to make a frozen section of the sample. The tissue is snap ...Download file to see next pagesRead More
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