Reliability of DNA cytometric S-phase analysis in surgical biopsies: Assessment of systematic and sampling errors and comparison between results obtained by image and flow cytometry

Three major parameters in DNA histograms that contribute to the reliability of S-phase analysis were evaluated. These parameters are (1) the extent of background in relation to the amount of S-phase cells (and the validity of its subtraction), (2) the size of the "free" S-phase range (S-free), and ( 3) the sampling error of cell counting. Tests in histograms obtained from s urgical biopsies by flow cytometry (FCM) showed that the background subtrac tion is reliable if the found S-phase fraction is higher than the fraction of background events in the histogram range of the cell population. The siz e of S-free was determined in computer-generated test histograms as a funct ion of variables such as the coefficient of variation (CV) and the DNA inde x (DI). To calculate the sampling error of cell counting above background a nd in S-free, a model was developed that was validated by experimental data . This error can serve as an indicator of the uncertainty in S-phase analys is. The poor correlation found between %S values measured by image cytometr y (ICM) and FCM in surgical biopsies was assigned to high uncertainty by lo w cell numbers in ICM histograms. A method is proposed to estimate quantita tively the reliability of S-phase analysis that can facilitate the interpre tation of results. Cytometry (Comm. Clin. Cytometry) 42:196-208, 2000. (C) 2000 Wiley-Liss, Inc.