Applications of Slide-Based Single Cell Cytometry by TissueFAXS in Histopathology

Background: Automated identification and cytometric quantification of molecular markers (antibodies, histological stains, genetic probes) on the single cell level in tissue sections by means of slidebased cytometry has become an essential tool in biomedical research and routine analysis in histopathology. TissueFAXS is the microscopic equivalent to flow cytometry – applicable to tissue sections, cell culture monolayers and cell smears/cytospin preparations and allows observer independent analysis of all types of cells in all kinds of tissue sections in-situ.

Technology & Method: The TissueFAXS system consists of a high-end motorized fluorescence microscope and sophisticated software for image analysis. The samples can be stained by means of immunohistochemistry or immunofluorescence. By using the nuclear marker (e.g. hematoxilin or DAPI) the location of the individual cells is identified and the systém quantifies the staining intensity in each color channel for each and every cell. It allows even to distinguish between and compare between nuclear and cytoplasmatic marker expression in the same sample. For each cell or cellular compartment up to 14 parameters are measured and stored in a database. Representation of cellular parameters may be obtained by dot plots and/or histograms in a FACS-like manner.

Results: Results presented come from several studies and comprise different applications of slidebased cytometry.(i) Phenotypic characterization of tissue infiltrating leukocytes in tumor biology(1), transplantation immunology(2) and autoimmune diseases(3) exemplifying the quantification of markers CD1a, CD3, CD4, CD8, CD11c, and CD68 in renal cell carcinoma, renal allograft rejection and atopic dermatitis. (ii) Signal transduction research in prostate cancer(4,5) and brain neuropathology(6) dealing with signal transducers and activators of transcription (STAT), and transforming growth factor beta. (iii) Quantification of proliferation marker Ki67 and Her2 in breast cancer.

Discussion: The data above demonstrate novel approaches in research and improved data transparency in diagnosis using slide-based cytometry. Observer-biased visual estimation in immunohistological analysis of tissue samples is replaced by observer independent measurements on the single-cell level, which is especially important in multi-center studies and set-up of clinical studies for the pharmaceutical industry.

1. Steiner GE et al. 2000, J Immunol Methods 237(1-2):39-5.
   
2. Kozakowski N, et al. 2009, Nephrology Dialysis Transplantation 24(6):1979-86.
   
3. Bangert C et al. 2010, Dermatology, 222(1):36-48.
   
4. Neuwirt H et al. 2009, Am J Pathol 174(5):1921-30.
   
5. Puhr M,et al. 2009, Cancer Res 69 (18), p. 7375-84.
   
6. Lohr J et al. 2011, Clinical Cancer Research, in press