title: Multi-parameter flow cytometric analysis with detection of the Ki67-Ag in paraffin embedded mammary carcinomas.
abstract: In the present study we describe a novel multiparameter flow cytometric (FCM) assay to estimate the fraction of cycling cells in epithelial tumors derived from fresh frozen as well as archival material. To this end, MCF-7 cells as well as a series of breast carcinomas (n = 10; fresh frozen as well as formalin fixed and paraffin embedded) were stained using a panel of different antibodies directed against the Ki67-Ag (DAKO/PC, MIB-1, Ki-S5, and poly-Ki67) for a 3-parameter cytokeratin/Ki67-Ag/DNA FCM analysis. Whereas all Ki67-Ag antibodies work equally well in the methanol fixed cell line, MIB-1 and Ki-S5 epitopes are retained in cell suspensions mechanically derived from fresh frozen tissue. Only antibody Ki-S5 shows specific nucleolar staining patterns in cell suspensions prepared by trypsin digestion of formalin fixed, paraffin embedded tissue sections. A good correlation was found between the fractions of Ki67-Ag-positive epithelial cells measured in cell suspensions derived from fresh frozen and the corresponding formalin fixed and paraffin embedded tumor samples. Furthermore, the fraction of Ki67-Ag-positive epithelial cells as determined by 3-parameter FCM correlated very well with the Ki67-Ag-labeling index in paraffin embedded tissue sections.
authors.affiliation: Department of Pathology, De Wever Hospital, Heerlen, The Netherlands.
authors.forename: M P
authors.initials: MP
authors.lastname: Leers
authors.forename: P H
authors.initials: PH
authors.lastname: Theunissen
authors.forename: F C
authors.initials: FC
authors.lastname: Ramaekers
authors.forename: B
authors.initials: B
authors.lastname: Schutte
mesh_terms: D000912:Antibodies, Neoplasm / Q000737:chemistry / Q000378:metabolism; D001943:Breast Neoplasms / Q000276:immunology / Q000378:metabolism*; D002469:Cell Separation; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D019394:Ki-67 Antigen / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D018613:Microscopy, Confocal; D008940:Mitotic Index; D016612:Paraffin Embedding; D014407:Tumor Cells, Cultured
chemical_list: D000912:Antibodies, Neoplasm; D019394:Ki-67 Antigen