The reproducibility of flow cytometric analyses in human tumors. Methodological aspects.

Various methodological aspects of flow cytometry were studied in material from endometrial carcinoma, ovarian tumors and bladder carcinoma. Measurements of identical samples on two different occasions gave an excellent correlation of the obtained DNA values, r = 0.997 and a good reproducibility of the S-phase rate, r = 0.87. In large tumors different DNA values were found in 5/36 cases when central and surface biopsies were compared (indicating tumor heterogeneity) which stresses the importance of multiple biopsies. The S-phase rate of the surface biopsies was generally higher. Comparing staining with ethidium bromide and DAPI, a good correspondence of ploidy determinations in human bladder tumors was found, provided that diploid cells from the normal tissue component were used as internal reference. When ethidium bromide staining was used, there was a good agreement between the values of tumor ploidy obtained by external standardization using lymphocytes and internal standardization using diploid tumor cells, respectively. In DAPI staining with lymphocytes as an external standard the tumor ploidy was systematically overestimated by about 10% due to suboptimal staining of lymphocytes after 3 hours. This difference decreased after 18 hours of staining. Determination of S-phase fraction showed a good correlation between DAPI and ethidium bromide stained bladder tumor samples (r = 0.86). Human lymphocytes as an external standard showed good reproducibility. In conclusion, flow cytometric measurements of the ploidy level and S-phase rate are highly reproducible provided that tumor heterogeneity is taken into account and proper preparation and standardization methods are used.