Preclinical applications of quantitative imaging cytometry to support drug discovery.

Preclinical drug development is actively involved in testing compounds to find cures or to manage the effects of disease, such as diabetes. Animal models, such as the Zucker diabetic fatty (ZDF) rat, are used to measure efficacy of candidate drugs. This animal model was selected because of its clinical and pathological similarities to diabetic human patients. A method using immunofluorescence and laser scanning cytometry (LSC) technology has been used to measure the development of diabetic phenotype in the ZDF rat during a 17-week time course. The expression levels of insulin, glucagon, voltage-dependent anion channel (VDAC), and Ki67 were quantified. Insulin and VDAC expression were reduced in the ZDF animals in comparison to the lean control rats, while no significant change was seen in glucagon and Ki67 expression at week 17. This information is useful in the design of studies to test experimental compounds in this model. Screening drug targets or biomarkers in tissue sections is another important activity in drug development. Tissue microarrays (TMAs) are composed of 60 or more tissue cores from humans or animal models and may contain healthy and/or diseased tissues. Antibodies against target proteins are applied to TMAs using routine immunohistochemical reagents and protocols. The protein expression across the cores, as labeled by immunohistochemistry, is measured using LSC technology. The process provides an efficient and cost-effective method for evaluating multiple targets in a large number of tissue samples. More recently, IHC and LSC have been taken to the next level to quantify biopharmaceutical drug and target co-localization in tissue sections.

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.