DNA flow cytometry of fresh and paraffin-embedded tissue using cytokeratin staining

DNA flow cytometry measurements were performed using cytokeratin as a second parameter to identify epithelial cells selectively in fresh and in archival paraffin samples of normal and adenocarcinoma tissues from breast and colon. Fresh specimens consisted of 22 adenocarcinomas of breast, 20 adenocarcinomas of colon, 16 control breast samples, and 13 control colon samples. Paraffin block specimens consisted of 22 adenocarcinomas of breast (the same as fresh samples), 20 adenocarcinomas of colon (the same as fresh samples), 37 control breast samples and 34 control colon samples. The average proportion of cytokeratin-positive cells per group ranged from 31 to 55% for fresh samples and from 14 to 34% for paraffin samples. For aneuploid cells populations of adenocarcinomas, which consist only of epithelial cells, the average percentage of cytokeratin-positive cells ranged from 60 to 72%. The technique gave satisfactory measurements of ploidy and of cell cycle data in both types of samples. Cell cycle measurements were less accurate than ploidy measurements in both types of samples, and multiple sampling will be required for adequate accuracy. The average S-phase fraction of cytokeratin-positive cells ranged from 6 to 15% for fresh specimens and from 11 to 20% for paraffin samples. Similar data were obtained for the proliferative index (G1 + S + G2 + M phases). The coefficients of variation were smaller for proliferative index than for S-phase fraction data, indicating greater accuracy. Paraffin data give higher cycling cell measurements than corresponding fresh data, so separate standardization of measurements may be required for fresh and for paraffin data.