Quantitative immunohistochemistry

Sompuram SR, Kodela V, Zhang K, et al. A novel quality control slide for quantitative immunohistochemistry testing. J. Histochem. Cytochem. 2002 50 1425-1434. 

Nadji M Quantitative immunohistochemistry of estrogen receptor in breast cancer. Much ado about nothing. Appl lmmunohistochem Mol. Morph. 2008 16 105-07. 

Zehntner SP, Chakravarty MM, Bolovan RJ et al (2008) Synergistic tissue counterstaining and image segmentation techniques for accurate, quantitative immunohistochemistry. J Histochem Cytochem 56 873-880... 

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Lindeman N, Waltregny D, Signoretti S, et al. Gene transcript quantitation by real-time RT-PCR in cells selected by immunohistochemistry-laser capture microdissection. Diagn. Mol. Pathol. 2002 11 187-192. 

Figure 5.3 Diagram depicts the further-designed studies to test our hypothesis with respect to standardization of immunohistochemistry based on the antigen retrieval technique exemplified in a multiple direction to draw a conclusion, (a) Periods of formalin fixation, (b) Variable delay of fixation, (c) Storage of FFPE tissue blocks or sections, (d) Variable thickness of FFPE tissue sections, (e) Other variable conditions of processing FFPE tissue blocks. The stereoscopic frame of a cube represents the reliable limitation of quantitative IFIC demonstrated by serial studies as recommended in the text. Reproduced with permission from Shi et al., J. Histochem. Cytochem. 2007 55 105-109.

It may be helpful to consider the following criteria when evaluating a proposed positive control for immunohistochemistry. A positive control is ideally (1) reproducible and quantitative, (2) available in unlimited quantities, (3) antigen-specific, (4) inexpensive, and thereby adaptable for mass production, and (5) stable over time. Judged against these criteria, current practice falls short of the ideal. Biopsy materials are variable, both from patient to patient and even within a single tumor sample. Biopsy or resection materials are also obviously not available in unlimited quantities. The need for (microtome) sectioning of each positive control also creates a labor cost that is often under-appreciated by many clinical IHC laboratories. 

When fresh or frozen tissue is used for proteomic analyses, the results cannot be related directly to the clinical course of diseases in a timely manner. Instead, researchers frequently reduce the number of interesting proteins to a manageable number and then attempt to use immunohistochemistry to understand the implications of proteomic changes in archival formalin-fixed, paraffin-embedded (FFPE) tissue for which the clinical course has been established.3 Unfortunately, immunohistochemistry is a semiquantitative pro-teomic method, and the choice of interesting proteins must occur without advance knowledge of the clinical course of the disease or the response to therapy. If routinely fixed and embedded archival tissues could be used for standard proteomic methods such as 2-D gel electrophoresis and mass spectrometry (MS), these powerful techniques could be used to both qualitatively and quantitatively analyze large numbers of tissues for which the clinical course has been established. However, analysis of archival FFPE tissues by... 

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... 

Presently, immunohistochemistry requires improvements in quality, reproducibility, speed, quantitation, and standardization. Some of these goals can be achieved by using computerized bar code-driven automatic immunostainers that automatically dispense reagents, control washing, mixing, and heating to optimize immunohistochemical reaction... 

Note that although immunohistochemistry is contributing significantly to clinical information, in the absence of quantitation, this method is subjective and prone to intra- and interobserver variations. To assure the reproducibility of histopathological results and then-correct evaluation, at least an assessment of the percentage of positive cells and both the staining pattern and intensity must be made. If available, computer-assisted image analysis... 

Another advantage of immunohistochemistry is that tissues of a small size (e.g., biopsies) can be used. This is important because it is better to detect tumors at an early stage, when they are small. The necessity of early detection cannot be overemphasized. Very small tumors and fine-needle aspirates cannot be used for biochemical assays. Although the DCC assay provides quantitative results, it does not take into account the relative amount of the connective tissue in the specimen, the presence of carcinoma in situ lesions, or normal ducts and lobules. These limitations are not encountered when using paraffin sections. In addition, immunohistochemistry allows the use of archival tissues when fresh tissues are not available. This method does not require any special, expensive equipment and can be carried out in any standard laboratory. 

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