Novel approaches for immunophenotyping by laser scanning cytometry (LSC)

LSC is a microscope-based technology. The principle of the instrument is that any specimen is immobilized on a microscope slide. Therefore the cells are not lost in a fluid stream but are kept on the slide and minimal specimens as low as 1.000 cells can be analyzed. Additionally cells are available for further analyses such as staining for another set of specific markers and re-analysis or cytological staining (H&E). This approach multiplies the information gained from a given sample. We have established an assay for immunophenotyping of peripheral blood leukocytes by LSC. Cells are prepared according to routine flow cytometry protocols with a first set of CD-antibodies and are fixed on microscope slides. As a stable trigger signal the nuclear DNA is stained by 7-aminoactinomycin-D. This guarantees that all nucleated cells and that only nucleated cells are included in the analysis, and many differentiate between lymphocytes and neutrophiles by staining intensity. After analysis cells are stained with a second set of CD-antibodies and analyzed again. This step can be repeated with a third set of CD-antigens. Since the location of the cells on the slide is fixed data from the analyses can be attributed to the same cell.