Dual-parameter flow cytometry of transitional cell carcinomas.Quantitation of DNA content and binding of carbohydrate ligands in cellular subpopulations

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Abstract
Quantitative DNA measurements and estimates of blood group-related carbohydrate antigen expression have been used as predictive parameters in transitional cell carciomas (Ca). To obtain an accurate quantitative characterization of cellular subpopulations on the basis of these parameters, the authors developed a dual-parameter flow cytometric method using a fluorescence-activated cell sorter. With this method single-cell suspensions from 26 transitional cell carcinomas were analyzed by means of propidium iodide (red fluorescence) as DNA ligand, and peanut agglutinin (PNA), wheat germ agglutinin (WGA), and anti-blood group A antibody (aBGA) as carbohydrate ligands. The latter ligands were visualized directly or indirectly by FITC (green fluorescence). The carbohydrate ligand binding was correlated to the DNA content of cell populations in the way that aneuploid populations showed a higher PNA binding (P < 0.0002) and a lower WGA (P < 0.01) and aBGA (P < 0.04) binding than did diploid cell populations. The binding of PNA to aneuploid populations could be further increased (P < 0.004) by neuraminidase treatments. Thus, aneuploid cells express both neuraminic acid substituted and unsubstituted PNA receptors. The carbohydrate ligand binding was cell cycle-dependent, as it was reduced (2-M phase. A low WGA (P < 0.004) or aBGA (P < 0.02) binding was correlated to tissue invasion. Immunohistochemistry with the carbohydrate ligands showed a good correlation between aBGA (P < 0.0005) and PNA (P < 0.004) binding to tumor cells and flow cytometric assay of these, as well as a correlation (P < 0.003) between cellular location of WGA receptors and flow cytometric assay of these. It seems that dual-parameter flow cytometry represents an important tool in the characterization of bladder tumors.