Doublet discrimination in DNA cell-cycle analysis

Robert P. Wersto, Francis J. Chrest, James F. Leary, Christa Morris, Mary Alice Stetler-Stevenson, Edward Gabrielson

Research output: Contribution to journalArticlepeer-review

126 Scopus citations


Differences in doublet analysis have the potential to alter DNA cell-cycle measurements. The techniques for doublet determination are often used interchangeably without regard for the complexity in cell shapes and sizes of biological specimens. G0/1 doublets were identified and quantitated using fluorescence height versus area and fluorescence width versus area pulse measurements, by enumerating the proportion of G2 + M cells that lack cyclin B1 immunoreactivity, and modeled in the DNA histograms by software algorithms. These techniques were tested on propidium iodide-stained whole epithelial cells or nuclei from asynchronous cultures, or after exposure to chemotherapeutic agents that induced cell-cycle arrest and were extended to human breast tumor specimens having DNA diploid patterns. G0/1 doublets were easily discernible from G2 + M singlets in cells or nuclei that are generally homogenous and spherical in shape. Doublet discrimination based on pulse processing or cyclin B1 measurements was nonconcordant in some nonspherical cell types and in cells following cell cycle arrest. Significant differences in G0/1 doublet estimates were observed in breast tumor specimens (n = 50), with estimates based on pulse width twice those of pulse height and nearly five times greater than computer estimates. Differences between techniques are attributed to difficulties in the separation of the boundaries between G0/1 doublets and G2 + M singlet populations in biologically heterogeneous specimens. To improve reproducibility and enhance standardization among laboratories performing cell cycle analysis in experimental cell systems and in human breast 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 comparison difficult. Published 2001 Wiley-Liss, Inc.

Original languageEnglish (US)
Pages (from-to)296-306
Number of pages11
JournalCommunications in Clinical Cytometry
Issue number5
StatePublished - Oct 15 2001
Externally publishedYes


  • Cylin B1
  • DNA cell cycle
  • Flow cytometry
  • G doublets
  • Modeling
  • Pulse height
  • Pulse width

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Biophysics
  • Hematology
  • Endocrinology
  • Cell Biology


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