Sendai virus-induced IFN-alpha production analysed by immunocytochemistry and computerized image analysis

IFN-alpha production in Sendai virus-stimulated human buffy coat cultures could readily be demonstrated in individual cells at a protein level by the use of a novel immuno-enzymatic staining procedure. A distinctive rounded, juxtanuclear staining pattern was generated in producer cells by the accumulation of the intracellularly synthesized IFN-alpha in the Golgi stacks. The technology is based upon acquiring a video image of stained monolayers of cells, viewed in a microscope by a colour camera, which then transfers binary images directly into a computer-controlled operating system. The characteristic appearance of the immunocytochemical 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 accurately and reproducibly register a range of 0.1-7.0% of the total cell population as IFN-alpha producing cells during the kinetic studies of the response. Congruent results were obtained with manual microscopy and image analysis concerning the assessment of the incidence of IFN-alpha producing cells in the total cell populations. All IFN-alpha producing cells expressed surface HLA-DR molecules and 95% of these cells belonged to the myelomonocytic lineage. The image analysis system provided, in contrast to conventional microscopy, an opportunity to assess and document differences of signal intensity and cell size of individual IFN-alpha producing as well as non-producing cells.