Immunohistochemistry Markers

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.

IHC principle

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.

Immunohistochemistry antibody

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.

Immunochemistry

The process of localizing proteins in cells of a tissue section using the principle of antigens in tissue binding to their respective antibodies is known as Immunohistochemistry (IHC). This technique is widely used for the diagnosis of cancer. The antibodies are tagged with color-producing dyes to make them easily visible. Horseradish peroxidase and phosphatase are the two commonly used color-producing tags. Different fluorophores like FITC are also used to tag the antibodies as an alternate method. This method is widely used in confocal laser scanning microscopy to visualize two interacting protein molecules. Adinocarcinomas, Hodgkin's disease, yolk sac tumors and hepatocellular carcinoma, Gastrointestinal Stromal Tumors (GIST) and prostate cancer are diagnosed by conducting immunohistochemistry using various antigens like Carcinoembryonic antigen, CD 15 & CD30 or Alpha fetoprotein or CD117 or prostate specific antigen accordingly. Direct and indirect methods are the two methods used for the immunohistochemical staining of antigens.

In the direct method, one labelled antibody is used for staining. The antibody directly binds to the antigen which is being stained. Involving only one labeled antibody (FITC conjugated antiserum), this direct method is also known as one-step staining method. Though this method is quick since it uses only one antibody, it is rarely used after the indirect method was introduced.

In the indirect method of immuno-histochemical staining, one antibody is used against the antigen which is being examined and a second labelled antibody is used against the first. The unlabelled primary antibody or the first layer reacts with tissue antigen and the second layer or the labelled secondary antibody reacts with the primary antibody. This indirect method is considered more sensitive, since there is good signal amplification noticed through many secondary antibody reactions with various antigenic sites on the primary antibody.

Histopathology

Histopathology involves study of diseased tissues thereby aiding diagnosis of tumors. Histopathological examination of tissues involves three stages - surgery, biopsy or autopsy.
After collecting the diseased tissues, they are first stabilized by placing in a fixative, to prevent decay. Formalin (10% formaldehyde in water) is the fixative used normally.

• The stabilized sample tissues are first transferred to a container where permitted reagents are allowed to act on the tissues.
  
• Concentrated ethanol helps to dehydrate the tissues. Toluene or Xylene is used after the above procedure to immerse the tissues followed by hot liquid paraffin wax. This process takes 12 to 16 hours, during which paraffin replaces the water in the tissue turning it soft and moist. Now the sample is ready for embedding.
  
• The tissue is transferred and set in a mold. During this process of embedding, additional paraffin is added to prepare a hard paraffin block.
  
• Microtome is used to section the embedded tissues into very thin sections of about 3-7 micrometer in thickness. These thin layers or sections are now ready for microscopic study.
  
• One or more pigments are used to stain the sections to view the details clearly. A combination of hematoxylin and eosin is used to stain the tissues. The cytoplasm pink is got by using eosin and nuclei blue by hematoxylin. Saffron, silver salts and some artificial dyes are the other compounds used to color tissue sections. For staining specific proteins, carbohydrates and liquids, antibodies are also used. The technique known as immunohistochemistry has helped scientists in the microscopic studies to identify different categories of cells specifically.
  
• A medically qualified expert or pathologist examines the histological slides microscopically.