Quantitation of Immunostaining

Accurate quantitation of antigens using immunohistochemistry depends upon a linear relationship between the amount of antigen and the intensity of immunoperoxidase-DAB reaction product as well as the percentage of stained cells. Variations in staining intensity will reflect the amount of antigen only if optimal preparatory procedures are used for example, oversaturation of the chromogen reaction may result in invalid quantitation. Therefore, optimal concentration of DAB should be determined by trials with DAB 

The system includes various software packages for different applications (De Cresce, 1986). It allows immunostained histological sections to be represented as digitized images from which the optical densities of the DAB reaction product over a specific cell part or component (e.g., nucleus) can be quantitated. Bacus et al. (1988) have successfully quantified the estrogen receptor content in human breast tumors. The data showed excellent sensitivity and specificity. 

A related approach was used by Ranefall et al. (1998) to quantify images of immunohistochemically stained cell nuclear Ki-67 antigen and cyclin A protein in bladder carcinoma tissue. They combined automatic, computerized image analysis with appropriate controls and reference material. This approach is superior to the automatic method without 

The image of the control cells serves as a standard control regarding image qualities such as illumination and color properties. Since control cells possess known characteristics regarding antigen expression to be examined in tissue sections, they serve as a means to control and standardize immunohistochemical data (Ranefall et al., 1998). 

Recently, an automatic color video image analysis system was developed to quantify antigen expression (androgen receptor) (Kim et al.,T999a). This system provides a linear relationship between the antigen content and mean optical density of the immunoperoxidase-substrate reaction product. Titration of antibody, concentration, and reaction duration of the substrate can be optimized with this system. The imaging hardware consists of a Zeiss microscope, a three-chip charge-coupled-device camera, a camera control board, and a Pentium-based personal computer. 

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