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Quantitative microscopical and confocal laser scanning microscopy for intermediate endpoint biomarkers in breast cancer: Potential and reproducibility
Author(s) -
Baak Jan P. A.,
MakkinkNombrado S.,
Tekola Paulos,
Bergers Elisabeth,
Beliën Jeroen A. M.,
Van Ginkel A. Hielke N.
Publication year - 1993
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.240531120
Subject(s) - reproducibility , confocal , flow cytometry , confocal laser scanning microscopy , confocal microscopy , coefficient of variation , biomedical engineering , pathology , cytometry , microscopy , biology , materials science , chemistry , microbiology and biotechnology , medicine , chromatography , optics , physics
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 ≥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 (≤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. Confocal laser scanning microscopy (CLSM) is a powerful new tool that can be used for 3‐dimensional (3‐D) microscopy and detects weakly fluorescent substances. We found CLSM useful to assess multidrug resistance (MDR) in tumor cells. The CLSM MDR method requires less than 50 cells, takes approximately 30 minutes, and currently is one of the most sensitive methods for assessing MDR. Moreover, 3‐D CLSM of thick (50 μm) breast cancer sections can result in realistic 3‐D views of whole tissue and also in accurate measurements of volume and shape factors of individual nuclei. As these features have proven to be strong prognostic factors in tumors of different sites, CLSM in combination with digital image processing (DIP) is a promising tool to accurately assess biomarkers in breast cancer patients. Cost‐benefit analysis shows that morphometry, stereology, and ICM have the best price‐performance ratios.