3-DIMENSIONAL CONFOCAL LASER-SCANNING DNA-PLOIDY CYTOMETRY IN THICK HISTOLOGICAL SECTIONS

DNA ploidy measurement by flow (FCM) or image cytometry (ICM) of singl e cell suspensions of solid tumour has prognostic value, but it would be a definite advantage if the assessment could be done on histologica l sections. However, this is usually not possible by means of standard ICM, due to the capping of nuclei in thin sections, or overlap in thi ck sections. Three-dimensional (3D) microscopy by means of confocal la ser scanning microscopy (CLSM) could solve this problem in theory but the results published so far are not very satisfactory. A new method h as been developed in which the DNA content of haploid (human testis sp ermatozoa), diploid, tetraploid, octaploid (human and rat liver and hu man spermatogonia), and near-triploid (human breast cancer) nuclei sta ined with YOYO-1 iodide has been measured by a newly developed 3D imag e cytometry method (3DICM) in 20 mu m thick histological sections. YOY O-1 iodide is a new highly sensitive, specific, stoichiometric, and st able fluorescent dye for DNA, DNA ploidy of a breast cancer which was near-triploid with FCM and ICM was also assessed,vith 3DICM in a tissu e section adjacent to the section used for FCM and ICM and the results were compared. The integrated 3DICM fluorescence intensity showed goo d 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 octaploi d (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 t he 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 indicts shelved excellent reproducibility between different observers (r=0.98). It is concluded that the 3DICM method used is accurate, reproducible, and clinically feasible in thic k histological sections. This is especially important in small lesions , or if the results of DNA ploidy measurement of single cell suspensio ns (by FCM) or imprints (by ICM) are inadequate.