High‐Resolution Multiparameter DNA Flow Cytometry for Accurate Ploidy Assessment and the Detection and Sorting of Tumor and Stromal Subpopulations from Paraffin‐Embedded Tissues

This article contains detailed protocols for the simultaneous flow cytometric identification of tumor cells and stromal cells and measurement of DNA content of formalin‐fixed, paraffin‐embedded (FFPE) tissues. The vimentin‐positive stromal cell fraction can be used as an internal reference for accurate DNA content assessments of FFPE carcinoma tissues. This allows clear detection of keratin‐positive tumor cells with a DNA index lower than 1.0 (near‐haploidy) and of keratin‐positive tumor cells with a DNA index close to 1.0 in overall DNA aneuploid samples, thus improving DNA ploidy assessment in FFPE carcinomas. Furthermore, the protocol is useful for studying molecular genetic alterations and intratumor heterogeneity in archival FFPE samples. Keratin‐positive tumor cell fractions can be sorted for further molecular genetic analysis, while DNA from the sorted vimentin‐positive stromal cells can serve as a reference when normal tissue of the patient is not available. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Multiparameter DNA content analysis of FFPE carcinomas Alternate Protocol 1: Immunocytochemistry for keratin and vimentin, and DNA labeling for blue and red excitation Alternate Protocol 2: Immunocytochemistry for keratin and vimentin, and DNA labeling for blue excitation Support Protocol: Sorting cell population from FFPE carcinomas