Immunohistochemistry, or IHC, has been used in the scientific community since 1941. It exploits the specificity of antibody binding to tissue-specific antigens; the method has historically been used to help identify and diagnose tumour cell populations. Disadvantageous aspects of IHC tend to stem from its lack of specificity, difficulty of interpretation and low percentage of reproducibility.
Photobleaching occurs when the fluorescent dyes used in IHC degrade due to overexposure. Oxygen radicals, which result from the photochemistry of fluorescence, react with the dyes and eventually destroy them. When viewing IHC prepared slides via confocal microscopy, the high-intensity laser can quickly render the fluorescent markers on the slide unusable after only a few viewings.
Because of the constantly shifting nature of scientific advance, many markers once thought to be exclusive to certain diseases may be shown to be nonexclusive---and thus unhelpful---indicators. For example, though the MIC2 antigen was originally thought to be specific to EWS-PNET (Ewing's sarcoma), later studies revealed that myriad other ailments also share the antigen.
Non-specificity occurs when the fluorescence in a given sample is not limited to any specific cell type (also called false positives). Non-specificity of fluorescent marker binding can not only waste samples, but if undetected, also lead to incorrect analysis.
Qualitative Nature of Analysis
IHC relies on qualitative observation. In order to conclude anything by IHC, one must score a sample manually, which involves deciding which cells in a given slide have fluoresced, and sometimes even counting them by hand. Deciding which cells have fluoresced can be especially problematic, as scoring results can vary greatly due to the quality of the dyes used, the length of time the sample cells have been exposed to the dye, as well as the judgment of scorer. As such, only a very low level scoring reproducibility exists.
- Molecular Station: Immunohistochemistry
- TATA Memorial Hospital: Utility of Ancillary Techniques in Pathologic Diagnosis of Pediatric Round Cell Tumors
- IVD Technology: Automated Image Analysis for Developing IVDs
- Laboratory Bulletin: Information from Rex Pathology Laboratory
- IHCWorld.com: Cell Biology Applications of Fluorescence Microscopy