Reliable DNA histogram interpretation: Number of nuclei requiring measurement with flow cytometry

OBJECTIVE: The reasons for conflicting prognostic results as to DNA ploidy and cell cycle variables (DNA index, percent S (%S) phase) may be found mainly at different levels of the flow cytometric methodology used. The present study concentrated on how many nuclei have to be measured with flow cytometry for reliable DNA histogram interpretation. STUDY DESIGN: Twenty- three samples of fresh frozen and 22 samples of paraffin-embedded material from different sites were used. For each sample, 5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 (x 1,000) events were cumulatively measured. The resulting DNA histograms were analyzed with the MultiCycle computer program using a highly reproducible interpretation protocol. RESULTS: No disagreements about DNA ploidy classification were found in samples with 10,000 or more events for the fresh frozen and 40,000 or more events for the paraffin-embedded material. Excluding DNA ploidy disagreements, the DNA index was stable in all cases. To obtain a %S-phase cell measurement within 20% of the reference value, at least 20,000 and 40,000 events were needed for, respectively, DNA diploid and DNA nondiploid cases for the fresh frozen material and, respectively, 40,000 and 50,000 events for the paraffin-embedded material. CONCLUSION: For reliable combined determination of DNA ploidy, DNA index and %S-phase cells, at least 40,000 and 50,000 events are necessary for fresh frozen and paraffin-embedded material, respectively.