THREE‐DIMENSIONAL CONFOCAL LASER SCANNING DNA PLOIDY CYTOMETRY IN THICK HISTOLOGICAL SECTIONS

Abstract

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 μm 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.