Doublet discrimination in DNA cell-cycle analysis

Differences in doublet analysis have the potential to alter DNA cell-cycle measurements. The techniques for doublet determination are often used inter changeably without regard for the complexity in cell shapes and sizes of bi ological specimens. G(0/1) doublets were identified and quantitated using fluorescence height v ersus area and fluorescence width versus area pulse measurements, by enumer ating the proportion of G(2) + M cells that lack cyclin BI immunoreactivity , and modeled in the DNA histograms by software algorithms. These technique s were tested on propidium iodide-stained whole epithelial cells or nuclei from asynchronous cultures, or after exposure to chemotherapeutic agents th at induced cell-cycle arrest and were extended to human breast tumor specim ens having DNA diploid patterns. G(0/1) doublets were easily discernible from G(2) + M singlets in cells or nuclei that are generally homogenous and spherical in shape. Doublet discri mination based on pulse processing or cyclin B1 measurements was nonconcord ant in some nonspherical cell types and in cells following cell cycle arres t. Significant differences in G(0/1) doublet estimates were observed in bre ast tumor specimens (n = 50), with estimates based on pulse width twice tho se of pulse height and nearly five times greater than computer estimates. D ifferences between techniques are attributed to difficulties in the separat ion of the boundaries between G(0/1) doublets and G(2) + M singlet populati ons in biologically heterogeneous specimens. To improve reproducibility and enhance standardization among laboratories p erforming cell cycle analysis in experimental cell systems and in human bre ast tumors, doublet discrimination analysis should best be accomplished by computer modeling. Shape and size heterogeneity of tumor and arrested cells using pulse-processing can lead to errors and make interlaboratory compari son difficult. Published 2001 Wiley-Liss, Inc.