In situ hybridization of immunoglobulin light chain mRNA on bone marrow trephines using biotinylated probes and the apaap method

A non-radioactive in situ hybridization method has been established for the detection of mRNA for the constant region of light chain immunoglobulin genes in routinely processed bone marrow trephines. The method utilizes a cocktail of biotinylated synthetic oligonucleotide probes to kappa or lambda mRNA. The method entails dewaxing of the paraffin-embedded sections, proteinase K treatment and overnight hybridization with the biotinylated probe. Detection of probe hybridization was performed by 2 immunocytochemical detection methods, utilizing either streptavidin-alkaline phosphatase or monoclonal anti-biotin followed by the alkaline phosphatase: anti-alkaline phosphatase (APAAP) labelling system. The new fuchsin/naphthol phosphate substrate yielded the strongest signal with specific localization of the hybridization signal to positive cells. Morphological preservation was excellent enabling both polyclonal and monoclonal plasma cells to be detected in bone marrow trephines. We conclude that this in situ hybridization method is no more difficult than standard immunohistochemical techniques and can be used in routine diagnostic laboratories.