Applications of laser scanning cytometry in immunohistochemistry and routine histopathology

Laser scanning cytometry (LSC) is a powerful tool for qualitative and quantitative analysis of tissue sections in preclinical drug development. LSC combines the strengths of flow cytometry with tissue architecture retention. This technology has been used predominantly with immunofluorescent techniques on cell culture and tissue sections, but recently LSC has shown promise in evaluating chromogenic immunohistochemistry (IHC) and histochemical products in paraffin-embedded and/or frozen tissue sections. Inverted light scatter measurements or a combination of inverted scatter and fluorescence allows automated determination of cell/nuclear counts (e.g., proliferation labeling indices), cell area (e.g., cellular hypertrophy), stromal elements, and labeling intensity (e.g., cytoplasmic/organellar proteins) in chromogen-labeled IHC or histochemical stained sections that correlates well with standard manual quantification methods. Segmentation with autofluorescence or dual immunolabeling facilitates capture of labeling data from specific cell populations. LSC evaluation of HE-stained sections is accomplished using autofluorescence/eosin fluorescence and inverse scatter. A standardized fluorescent approach with archivability, a lack of fluorescence quenching (photobleaching), and amenability to evaluation of multiple markers in a section has been demonstrated using Qdot nanocrystals. Examples of LSC use in chromogenic IHC, routine histopathology, and Qdot labeling will be reviewed, and advantages and disadvantages of this technology will be discussed.