Image analysis versus flow cytometry for DNA ploidy quantitation of solid tumors: a comparison of six methods of sample preparation

Abstract

With the availability of user-friendly interactive image analysis instruments for DNA analysis, there is a growing need for comparison with the established methodology of flow cytometry. We have compared the results of DNA ploidy quantitation in 12 solid tumors prepared by six different techniques of sample preparation: flow cytometry of fresh cell suspensions and of nuclei isolated from formalin-fixed, paraffin-embedded tissue; and image analysis of touch preparations, of disaggregated cells from paraffin-embedded tissue as well as of 3- and 7-microns-thick tissue sections. Complete agreement in DNA ploidy results obtained by the six methods was found in six out of 12 solid tumors. Image analysis of touch preparations detected most tetraploid and multiple aneuploid peaks. Sections of 7-microns-thick tissue gave better histogram quality than 3-microns-thick sections, however tetraploid peaks were not resolved in one case. Image analysis of disaggregated paraffin-embedded tumor showed comparable ploidy to fresh touch preparations in seven out of 12 cases, the discrepancies being due to loss of tetraploid or multiple aneuploid peaks. Flow cytometry gave good histograms, but tetraploid and multiple aneuploid peaks were occasionally not detected. Each method presents advantages and disadvantages. Flow cytometry and image analysis are complementary methods for DNA quantitation, and more than one method may be necessary to confirm the DNA content of solid tumors.