Comparison of DNA histograms by standard flow cytometry and image cytometry on sections in Barrett's adenocarcinoma

BACKGROUND: The purpose of this study was to compare DNA histograms obtained by standard flow cytometry (FC) and high fidelity image cytometry on sections (ICS) in normal gastrointestinal mucosa and Barrett's adenocarcinoma (BAC). METHODS: Archival formalin-fixed paraffin-embedded tissue blocks of 10 normal controls from 10 subjects and 42 BAC tissues from 17 patients were examined. DNA FC was performed using standard techniques and ICS was carried out by Automated Cellular Imaging System (ACIS). DNA ploidy histograms were classified into diploid with peak DNA index (DI) at 0.9–1.1, and aneuploid with peak DI > 1.1. DI values of aneuploid peaks were determined. Additionally, for DNA ICS, heterogeneity index (HI) representing DNA content heterogeneity, and histograms containing cells with DI > G2 were also identified. RESULTS: All control samples were diploid by both FC and ICS analyses. In BAC, FC showed diploid peaks in 29%, diploid peaks with additional aneuploid or tetraploid peaks in 57%, and 14% of the samples, respectively. In contrast, ICS showed aneuploid peaks in all the cases with peak DI > 1.25; 37 cases had peak DI between 1.25 and 2.25; and 5 cases had peak DI > 2.25. HI values (mean ± SD) were 11.3 ± 1.1 in controls and 32.4 ± 8.5 in BAC (p < 0.05). Controls had no G2 exceeding cells. However, 19/37 (51%) of the cases with primary peak DI < 2.25 had cells exceeding 9N. CONCLUSION: ICS detects DNA aneuploidy in all BAC samples while FC missed the diagnosis of aneuploidy in 29%. In addition, ICS provides more information on HI and G2 exceeding rates.