Immunohistochemicalmarkers (IHC markers) act as precursors for the identification of cancers or tumors in the body. They are made up of monoclonal antibodies which determine the target-specific proteins present in the respective tissue. These antibodies adhere to the proteins which are indicated by using stains. The diagnostic approach by using immunohistochemicals effectively identifies the type of protein to determine the histological changes of a specific tumor or cancer when identified. It also helps in the identification and categorization of homogeneous malignant tumors.
Specimen preparation for IHC
In the IHC technique, signal amplification and visualization is generally used and hence the selection of appropriate antibodies to target the antigens is very important. In addition to this the cell morphology, tissue design and antigenicity is also taken in to consideration. The tissues obtained are preserved carefully to prevent cellular damage and tissue morphology. Tissue fixation is done using formaldehyde as it enables proper adherence in preventing the onset of target antigen masking. In order to avoid the nonspecific protein site binding by the antibodies, the specimens are kept in buffer solutions such as bovine serum albumin, serum and non-fat milk in dried form.
The important principle administered in the immunohistochemical technique is based on the antigen-antibody reaction. The monoclonal antibody administered in the process binds to the tissue antigen for a complex with the help of secondary enzyme-conjugated antibody in the presence of a substrate and a chromogen for colored identification. The enzyme forms deposits at the site of antigen and antibody complex which can be determined by using computerized mechanisms to identify the locus of the tumor or cancer if present through protein determination.
The detection antibody plays a very significant role in the process of IHC technique. The categories of antibodies used vary from monoclonal to polyclonal which is determined by the type of diagnosis. Monoclonal antibodies identify single epitopes pertaining to linear or conformational in origin. The specificity of monoclonal antibody administration is seen in lower backgrounds and of polyclonal antibodies is seen in high backgrounds. Monoclonal antibody in tissues is administered overnight with a concentration of 5-25 micrograms/ml at 4°c whereas polyclonal antibodies are administered in the concentration of 1.7-15 micrograms/ml at 4°c.
The concentration of monoclonal antibodies and polyclonal antibodies for cells is similar to tissues but the duration of time is one hour at room temperature. Despite many advantages, monoclonal antibodies have disadvantages with respect to vulnerability in epitope masking and polyclonal antibodies have accumulation of heterogeneous population. The results of IHC antibody administration is carried on using various combinations.
The following proteins are identified in various tissues by using the antibodies. 1. Vimentin is a filament predominantly expressed in the connective tissue tumors and melanomas.
2. Leukocyte common antigen which is significantly found in leukemia and lymphomas.
3. CD 20 which is found in B-lymphocytes and CD3 found in T-lymphocytes.
4. Smooth muscle actin an important intermediate filament - This marker is usually expressed in myofibroblastic and myoepethelial cell tumors.
5. Cytokeratins are also used in the detection of adenocarcinomas and other carcinoma. The combinations of cytokeratins include both low molecular and high molecular weight compounds.
Complications in the IHC technique
IHC markers are very important in the determination of target therapies for cancers and tumors. The most common problem encountered in the immunohistochemistry technique is the background staining. Background staining is related to unwanted specific staining when mediated by interactions of antibodies and their respective epitopes and also nonspecific staining for all other interactions. The incidences of background staining are generally blocked by using reagents such as peroxidases.
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Bibliography / Reference
Collection of Pages - Last revised Date: September 30, 2023