Studies of amplification methods in immunohistochemistry
The aim of this project was to compare and contrast the results of three related methodologies which are of particular relevance or potential within our department -. a manually performed streptAvidin Biotin Complex (sABC/ HRP) method which for many years has served as our 'standard' immunocytochemical method; an integrated semi-automated systems from DAKO based on the TechMate 500 immunostainer and the ChemMate reagent package;. a manually performed tyramide amplification method regarded as amongst the most sensitive immunocytochemical method currently available. Antigen retrieval investigating differing durations of exposure to the proteolytic enzyme trypsin, and heat mediated antigen retrieval using citrate buffer pH6.0 or DAKO high pH antigen retrieval solution were also included as parameters for investigation. To this aim a panel of eleven specific antibodies were assessed, each of which had previously displayed individual qualities worthy of investigation -. PGP9.5, CD30, oestrogen receptor, CD3 polyclonal, CD2, CD3 monoclonal, CD4, cyclin D1, kappa immunoglobulin light chain, lambda immunoglobulin light chain and IgD immunoglobulin heavy chain. The results of this work demonstrate that each of the systems investigated has its individual merits -. The manual sABC/ HRP method is the least complicated and expensive of the methods to perform. There are significant disadvantages in its used however. Being a manual method there are many steps where individual variation in working practices within a laboratory can and do affect results. The TechMate/ ChemMate system is a more sensitive system than the manual sABC/ HRP method also providing a cleaner end result and allowing higher dilutions of primary antibody to be employed. Tyramide amplification is the most sensitive system evaluated, the antibodies in this work generally demonstrated immunostaining at higher dilutions than the other methods tested although some, in particular those directed against immunoglobulin light chains, operated better under the ChemMate/ TechMate system. On occasion specific positive results were obtained which none of the other methods tested could achieve with certain antibodies, CD3 monoclonal and to a lesser extent CD2.