PRINCIPLES OF PLOIDY ANALYSIS BY STATIC CYTOMETRY

Aims-To examine the basic assumptions made during DNA ploidy analysis of histological sections with an aim to eliminate methodological error s that have lead to conflicting results with this technique. Methods-A rat Liver imprint and histological sections together with sections an d whole nuclei cytospins of human breast tumour biopsy specimens were stained with azure A Schiff's reagent and used to investigate the effe cts of computer imaging, histological section thickness, nuclear volum e, and shape corrections. Results-The rat liver imprint demonstrated a linear relation between mean nuclear transmittance and nuclear area f or each of the three (2C, 4C, 8C) hepatocyte clusters. This finding wa s used to produce similar, proportional integrated optical density (IO D) measurements from rat liver sections by selecting only nuclei that were sectioned through their centres, as assumed by the mathematics fo r volume correction. The limitations of computer imaging necessitated an edge (glare) correction for each nucleus so that nuclei of differen t sizes could be analysed. Shape correction was required to analyse nu clei of different morphologies. Normal human lymphocytes, squamous epi thelium and fibroblasts were all measured with similar IODs. DNA ploid y values obtained from 7 mu m human breast tumour sections (using lymp hocytes as controls) correlated well with those obtained from the whol e nuclei cytospins from the same tissue blocks. Conclusions-With an im proved understanding of the theoretical and technical aspects of ploid y analysis of tissue sections, reproducible and consistent results are possible. These results can be integrated into routine histopathology investigations alongside immunohistochemistry and molecular diagnosti c techniques.