SENDAI VIRUS-INDUCED IFN-ALPHA PRODUCTION ANALYZED BY IMMUNOCYTOCHEMISTRY AND COMPUTERIZED IMAGE-ANALYSIS

IFN-alpha production in Sendai virus-stimulated human buffy coat cultu res could readily be demonstrated in individual cells at a protein lev el by the use of a novel immuno-enzymatic staining procedure. A distin ctive rounded, juxtanuclear staining pattern was generated in producer cells by the accumulation of the intracellularly synthesized IFN-alph a in the Golgi stacks. The technology is based upon acquiring a video image of stained monolayers of cells, viewed in a microscope by a colo ur camera, which then transfers binary images directly into a computer -controlled operating system. The characteristic appearance of the imm unocytochemical staining enabled a computerized image-analysis system to measure IFN-alpha producing cells based on defined criteria set for morphology, intensity, colour and size. The automated system could ac curately and reproducibly register a range of 0.1-7.0% of the total ce ll population as IFN-alpha producing cells during the kinetic studies of the response. Congruent results were obtained with manual microscop y and image analysis concerning the assessment of the incidence of IFN -alpha producing cells in the total cell populations. All IFN-alpha pr oducing cells expressed surface HLA-DR molecules and 95% of these cell s belonged to the myelomonocytic lineage. The image analysis system pr ovided, in contrast to conventional microscopy, an opportunity to asse ss and document differences of signal intensity and cell size of indiv idual IFN-alpha producing as well as non-producing cells.