DNA measurement errors with a scanning microdensitometer in cytologic and histologic samples of breast cancers

The influence of various DNA measurement errors using a commercially available scanning microdensitometer was evaluated on Feulgen-stained cytologic and histologic samples prepared from paraffin blocks containing invasive ductal breast cancers. The overall average total measurement error was 5.5% for the cytologic specimens and 10.9% for the 4 μm histologic sections. Components of the error included microscopic adjustment variation and focussing errors (3.5% and 1.1%, respectively, for both cytologic and histologic samples) and background intensity estimation errors (3.0% for the cytologic samples and 10.0% for the histologic samples). Measurements of the integrated optical density had a minimal error of 0.5% and an average error of 1.0%. Limitations due to the histologic architecture and/or heterogeneous cell population gave rise to large differences in the selection of nuclei when differently sized scanning masks were used. To improve the reproducibility, masks used should be based on the individual cell size, and background intensity values should be carefully estimated in the vicinity of the selected cells. Overall, the cytologic tumor samples were preferable to the histologic samples for static DNA measurements. It was easier to select cells suitable for measurement in the cytologic samples, and the cytologic measurements were less time consuming and produced a smaller measurement error.