Quantitative microscopical and confocal laser scanning microscopy for intermediate endpoint biomarkers in breast...

Diagnostic quantitative pathological (QP) determinations are increasingly used in our hospital. The number of requests for QP for reference materials is rising rapidly. This is understandable; quantitative assessments have a strong prognostic value and can be very reproducible, depending on the care taken with a number of factors including cell and tissue processing, application of the appropriate stains, and the measurement protocol used. As to the latter, systematic random sampling gives the best intra- and interobserver agreement (with correlation coefficients between observers for certain features > or = 0.94). Flow cytometric determinations are often regarded as more reproducible than interactive morphometry due to the high speed of the assessments, the large number of objects measured per specimen, and the lack of observer interaction. Indeed, flow cytometrically assessed DNA ploidy is very reproducible, even though the % S-phase fraction is much more variable. Unlike image cytometry (ICM), visual inspection of cells is not easily accomplished with flow cytometry (FCM). With ICM, the fully automated measurement of DNA in thousands of cells is possible in 3-5 minutes, with a very low coefficient of variation (< or = 2% for the diploid and tetraploid peak of liver cell nuclei). ICM also allows measurement of texture features. However, quantitative immunohisto/cytochemical determinations may not always be as reproducible as sometimes believed. Recently, we found large variations in the measurements, made by a commercially available image processing instrument, of the estrogen and progesterone receptors, Ki-67, cathepsin D, and neu protein overexpression in breast cancer.(ABSTRACT TRUNCATED AT 250 WORDS)