Immunohistochemistry has become a generally accepted and widely used auxiliary method of diagnostic pathology, including the pathology of bone tumors and tumor like lesions.
This method has emerged as a diagnostically useful technique because of the development of highly specific antibodies and the invention of sensitive immune and enzymatic detection systems. The fluorescence detection methods are more often used in investigative studies and are rarely sued in diagnostic pathology.
Basis
The Immunohistochemical method is based on binding of a specific cellular or extracellular substance by the antibody, with subsequent visualization of the bound antibody by a color-based detection system. Subsequent use of a counterstain such as hematoxyline or toluidine blue enables the precise microscopic localization of a positive reaction in various components of the tissue.
The avidin-biotin and peroxidased-based detection systems are most frequently used.
In formalin-fixed, paraffin-embedded tissue, the so-called antigen-retrieval techniques are important steps in increasing the sensitivity of the test. The goal of this step is to expose the epitopes of the studies antigen and to facilitate antigen antibody binding.
The antigen-retrieval techniques are simple and typically include limited digestion with proteolytic enzymes, microwave treatment, or both.
At this level of advancement, Immunohistochemistry provides consistent results with most fixatives. Satisfactory results can be obtained in decalcified tissue or even on decolorized, previously stained microscopic sections. It can also be used in most cytologic preparations.
Constant daily comparison of the immunohistochemical results with external and internal positive and negative controls helps prevent misinterpretation of ambiguous results. The pitfalls of immunohistochemistry are in general caused by false-negative or false-positive results.
False-negative results:
1 The epitopes of antigens in the tissue are lost because of inadequate fixation. The amount of antigen is reduced by degradation and cannot be detected by immunohistochemistry.
2 The antigen are misplaced from the specific tissue or cellular location because of diffusion. This is most frequently observed in reference to endothelial antigens such as factor VIII-related antigen.
3 The antibody is inappropriately used (too low a concentration) or destroyed, or its affinity for the antigen is inadequate.
4 The components of the detection system are inadequate or are inappropriately used.
False-positive Results:
1 Cross-reactivity (lack of specificity) of the antibody with other antigens or its nonspecific binding to the tissue.
2 Nonspecific color reaction caused by the presence of unblocked endogenous peroxidase.
3 Nonspecific binding of detection system components, such as the avidin-biotin complex, to the tissue (This is typically caused by excessive use of detection system components).
5 Misplacement of the antigen from normal tissue that is subsequently absorbed or phagocytized by tumor cells False-positive results are in fact more misleading than false-negative results and probably occur more frequently.
immunohistochemistry is a powerful tool used to provide diagnostically valuable information on the histogenesis and differentiation of cells. Similar to other auxiliary techniques, the immunophenotypic profiles of cells are not used to distinguish among benign, malignant, and reactive conditions.
The number of antibodies with potential diagnostic applications is huge, and new antibodies are constantly being developed. The specific applications of immunohistochemical stains and the so-called immunophenotypic features of bone tumors are provided in the sections on special techniques that accompany the discussion of each specific bone tumors.
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