Analysis of Tissue Imprints by Scanning Laser Cytometry

unit 7.22

The diagnosis of solid tumors is based primarily on the histological analysis of formaldehyde-fixed and paraffin-embedded material. The importance of determining the biological aspects of tumors is, however, growing for the clinical practice. This also necessitates quantitative measurements obtained by well-established cytometric methodology. A major advantage of slide-based cytometry (e.g., scanning laser cytometry; unit 2.10) in contrast to flow cytometry is the opportunity to morphologically evaluate cell populations according to immunophenotypic characteristics following microscopic repositioning. The use of tissue imprints for this purpose significantly simplifies measurements in solid-tumor cells.

The immunolabeling strategies described here are intended for the fast analysis of total nuclear DNA content and other functional aspects of tumor cells in cytological specimens using a laser scanning cytometer (LSC). The Basic Protocol describes the rapid assessment of tumor cell ploidy in any type of solid tumor using CD45-positive (CD45+) tissue leukocytes as internal reference cells with normal (2N) DNA content. An Alternate Protocol is provided for the quantitative measurement of nuclear antigen expression, such as estrogen and progesterone receptors, as well as the proliferation-related MIB1 antigen.

The methods are simple and rapid and are designed to perform with success despite the variation in quality of routine surgical material. Therefore, two-color analyses of DNA content (propidium iodide; PI) and an immunological marker (fluorescein isothiocyanate; FITC) are preferred. To keep the number of slides as low as possible, multiple areas within the same slide can be designated with a grease pencil and processed with different primary antibodies. Neighboring areas on one slide can then be measured separately in the LSC. The DNA content distribution, which is the same in all the measurements, serves to identify the respective cell populations in tumors with aneuploidy.

The methods rely on the analysis of tissue imprints (Support Protocols 1 and 2), but the labeling can be carried out on other freshly prepared cytological material, including smears obtained after scratching the tissue surface or following cell isolation procedures (unit 5.2). In this case, isolated cells from solid tumors should be deposited on slides by cytocentrifugation (2000 rpm, 7 min). To obtain large areas (<240 mm2), a Hettich 1640 cytocentrifuge (Andreas Hettich) or similar instrument should be used.

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