Determination Of Ploidy In Carcinomas By Lsc Of Fineneedle Aspirate Biopsies

FNABs are taken using a 20-ml syringe connected to a 27-G (or smaller-diameter; i.e., < 0.4-mm) needle. The material is resuspended in phosphate-buffered saline (PBS) and erythrocytes are lysed. The cells are then mounted on conventional glass microscope slides, air dried, fixed, and stored in ethanol. For analysis, cells are stained for cytokeratin by immunofluorescence and for DNA by incubation with propidium iodide (PI). Analysis in the LSC is then triggered on nuclear fluorescence, and a variety of parameters are determined for the fluorescence of cytokeratin and DNA and for the forward light scatter (FS). For the interpretation of the data, the slide is counterstained with hematoxylin/eosin (H&E) and the DNA content of the cytokeratin-negative leukocytes is set to 1.0. The DNA content of the cytokeratin-positive tumor cells is then determined and the DNA index (DI) of the tumor is calculated as the ratio of tumor DNA content to leukocyte DNA content; in order to obtain more commonly used ploidy values, the DI is multiplied by the factor 2 (i.e., DI 1.00 = 2c). The morphology of the different cell types (leukocytes, tumor cells) is verified and documented with the built-in CCD camera, or any other camera, by taking micrographs after relocalization of single cells in the LSC.

Materials

Subject with solid tumor of interest PBS with 1% and 0.5% BSA (see recipe)

Erythrocyte lysing solution: ammonium chloride lysing solution (appendix 2A) or commercial lysing product (e.g., FACS Lysing Solution from Becton-Dickinson) 70% ethanol

Phosphate-buffered saline (PBS; appendix 2a)

FITC-conjugated anti-cytokeratin antibody and negative-control isotype (both mouse lgGs; Dako) Anti-FITC antibody conjugated to Alexa Green (Molecular Probes) PBS with 0.5% BSA, 50 |g/ml PI, and 100 |g/ml RNase (see recipe) Glycerol/PBS/PI (see recipe)

unit 7.20

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