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

Critical Evaluation of the Use of Indirect Immunocytochemical Techniques in Diagnostic Pathology Introduction: 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 frozen by quenching in liquid nitrogen. Then the frozen sample is attached to cryostat chuck, and the sample is placed in the cryostat, and after allowing time, sections are made. Antigen Retrieval: Formalin fixation has the disadvantage of building protein cross links. Pretreatment with antigen retrieval reagents break the protein cross links to expose antigenic sites, and this improves antigen detection. Enzymatic method uses enzymatic digestion by proteolytic induced epitope retrieval or PIER. The other method is heat induced that involves application of heat to formalin-fixed paraffin tissue sections in a retrieval solution. For this, microwave oven or pressure cooker methods are used. The heating length is 20 minutes followed by a cooling for 20 minutes. A citrate buffer at pH 6.0 gives satisfactory retrieval for most preparations. Types of Labels: The final sensitivity of the method is dependent on labeling the primary antibody. Nonenzymatic labels use fluorescent labels. Enzymatic labeling uses histochemical enzymes and their substrates to label an antibody. Affinity labeling method uses avidin-biotin labeling. Probe labeling uses different radioactive isotopes to detect an antigen. Non radioactive probe labeling uses nonisotopes to label the antibody. The antibodies that are used for labeling are of immunoglobulin G class, some may be IgM class. Examples: Protein A method : Protein A is a major cell wall protein of Staphylococcus aureus. It has a very high affinity for Fc component of the immunoglobulin. It can be conjugated to various labels, such as, fluorescein, gold, alkaline phosphatase, and peroxidase without impairment of function. Advantages and Disadvantages of Indirect IHC: The complete assay in indirect IHC involves incubation with unlabeled primary antibody, washing, incubation with secondary antibody followed by washing and detection. The labeled secondary antibody allows for signal amplification compared with the direct method, and sensitivity is increased, allows for commercial development and standardization of these detection reagents. Background staining associated with polyvalent secondary antibodies is a problem. Specificity and Sensitivity: This is more specific and sensitive in comparison to direct techniques, specially when applied to the intracellular structures or inclusions. Use in Diagnostic Pathology: The most widespread use is to supplement the morphologic criteria in determining the appropriate classification of an undifferentiated neoplasm. Other than this, this technique is used in analytical assays, detection of infectious agents, determining prognostic markers including hormone receptors, oncogene products, or proliferation markers. Immunogold Techniques: Labeling with colloidal gold particles that may be used in different particle sizes to carry out multiple staining at the electron microscope label, most easily by direct labeling of several first layer antibodies. Conclusions: Immunohistochemistry is a novel approach to use a natural reaction between two macromolecules, an antigen and an antibody, outside the body to make a unique pathologic diagnostic tool for present-day medicine. Read More