Multiparametric evaluation of flow cytometric synthesis phase fraction determination in dual-labelled breast carcinomas

Multiparametric, two-color DNA and cell cycle analyses were performed on 112 consecutive mechanically dissociated, ethanol-fixed breast carcinomas using a dual-label method with monoclonal antibodies (CAM 5.2) to cytokeratin (CK) and leukocyte common antigen (LCA) with propidium iodide (PI) staining. There was marked intertumoral variation of CK-positive (range, 3-87%; mean, 40%) and LCA-positive (range, 1-28%; mean, 6.5%) events in DNA histograms. Approximately 70% of DNA aneuploid cells were CK positive. CAM 5.2-stained (avidin-biotin technique) Cytospin preparations correlated with flow cytometric (FCM) detection of CK-positive cells in 15/21 (71%) cases. In each discrepant case, FCM detected greater numbers of CK-positive cells. Cytospin controls of tumor suspensions revealed that cytoplasmic loss was the major cause of decreased CK staining. Synthesis phase fraction (SPF) calculation from CK-gated histograms resulted in kinetic indices (mean ungated, 12.3%, vs. mean CK-gated, 16.8%; P <.01) with improved statistical correlations with tumor grade and estrogen receptor (ER) status. Differences between ungated vs. CK-gated SPF were greatest in cases having <20% CK-positive events (P <.05). Cases with lower CK staining events generally had higher SPF and were more often high grade (below median CK staining, 61% high grade, vs. above median CK staining, 31% high grade) and ER-negative (below median CK staining, 55% ER negative, vs. above median CK staining, 12% ER negative). We conclude: (1) multiparametric, two-color DNA analysis allows measurement of the epithelial cell proliferative fraction excluding contaminating non-epithelial cell components, (2) cytoplasmic loss from either mechanical dissociation procedure or postexcision autolysis accounts for limited epithelial representation in some CK-gated histograms, (3) FCM is extremely sensitive in the detection of cells with small cytoplasmic remnants attached to nuclei, and (4) tumors with rapidly cycling cell populations are more susceptible to cytoplasmic loss in this whole cell method of analysis.