Tekola P, Baak J P, van Ginkel H A, Belien J A, van Diest P J, Broeckaert M A, Schuurmans L T
Department of Pathology, Free University, Amsterdam, The Netherlands.
J Pathol. 1996 Oct;180(2):214-22. doi: 10.1002/(SICI)1096-9896(199610)180:2<214::AID-PATH633>3.0.CO;2-P.
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.
通过流式细胞术（FCM）或图像细胞术（ICM）对实体瘤单细胞悬液进行DNA倍体测量具有预后价值，但如果能在组织切片上进行评估将具有明显优势。然而，由于薄切片中细胞核的封端或厚切片中的重叠，通过标准ICM通常无法做到这一点。理论上，利用共聚焦激光扫描显微镜（CLSM）进行的三维（3D）显微镜检查可以解决这个问题，但迄今为止公布的结果并不十分令人满意。已经开发出一种新方法，通过一种新开发的三维图像细胞术方法（3DICM），在20微米厚的组织切片中测量用YOYO-1碘化物染色的单倍体（人类睾丸精子）、二倍体、四倍体、八倍体（人类和大鼠肝脏以及人类精原细胞）和近三倍体（人类乳腺癌）细胞核的DNA含量。YOYO-1碘化物是一种新型的对DNA高度敏感、特异、化学计量且稳定的荧光染料。对一个通过FCM和ICM检测为近三倍体的乳腺癌，在与用于FCM和ICM检测的切片相邻的组织切片中，也用3DICM进行了评估，并比较了结果。3DICM的积分荧光强度与所有分析细胞核的实际DNA含量显示出良好的线性关系（r = 0.99）。在人体组织中，单倍体（n = 12）、二倍体（n = 63）、三倍体（n = 13）、四倍体（n = 12）和八倍体（n = 3）倍体分布的3DICM变异系数分别为5.1%、6.6%、4.2%、4.0%和0.6%（n = 细胞核数量）。对于大鼠肝脏，二倍体（n = 21）、四倍体（n = 31）和八倍体（n = 3）峰的CV分别为6.7%、4.8%和1.6%。对具有不同DNA指数的细胞核进行重复的“盲法”测量显示，不同观察者之间具有出色的可重复性（r = 0.98）。结论是，所使用的3DICM方法在厚组织切片中准确、可重复且临床可行。这在小病变中尤为重要，或者当单细胞悬液（通过FCM）或印片（通过ICM）的DNA倍体测量结果不充分时。
