DNA ploidy measurement by flow (FCM) or image cytometry (ICM) of single cell
suspensions of solid tumour has prognostic value, but it would be a definite advantage if the assessment could be done on histological sections. However, this is usually not possible by means of standard ICM, due to the capping of nuclei in thin sections, or overlap in thick sections. Three-dimensional (3D) microscopy by means of confocal
laser scanning microscopy (CLSM) could solve this problem in theory but the results published so far are not very satisfactory. A new method has been developed in which the
DNA content of haploid (human testis spermatozoa), diploid,
tetraploid, octaploid (human and rat liver and human spermatogonia), and near-
triploid (human
breast cancer) nuclei stained with
YOYO-1 iodide has been measured by a newly developed 3D image cytometry method (3DICM) in 20 microns thick histological sections.
YOYO-1 iodide is a new highly sensitive, specific, stoichiometric, and stable
fluorescent dye for
DNA.
DNA ploidy of a
breast cancer which was near-
triploid with FCM and ICM was also assessed with 3DICM in a tissue section adjacent to the section used for FCM and ICM and the results were compared. The integrated 3DICM fluorescence intensity showed good linearity (r = 0.99) with the real
DNA content of all nuclei analysed. In human tissue, the coefficient of variation of 3DICM for haploid (n = 12), diploid (n = 63),
triploid (n = 13),
tetraploid (n = 12), and octaploid (n = 3) ploidy distributions was 5.1, 6.6, 4.2, 4.0, and 0.6 per cent, respectively (n = the number of nuclei). For the rat liver, the CV of the diploid (n = 21),
tetraploid (n = 31), and octaploid (n = 3) peaks was 6.7, 4.8, and 1.6 per cent, respectively. Repeated "blind' measurements of nuclei with different
DNA indices showed excellent reproducibility between different observers (r = 0.98). It is concluded that the 3DICM method used is accurate, reproducible, and clinically feasible in thick histological sections. This is especially important in small lesions, or if the results of
DNA ploidy measurement of single cell
suspensions (by FCM) or imprints (by ICM) are inadequate.