Leif R C, Nordqvist S, Clay S, Cayer M, Ingram D, Cameron B F, Bobbitt D, Gaddis R, Leif S B, Cabanas A
J Histochem Cytochem. 1977 Jul;25(7):525-37. doi: 10.1177/25.7.894001.
The dissociation of cervical cell suspensions after various chemical and enzymatic treatments was monitored by using the Centrifugal Cytology rotor to produce glutaraldehyde-fixed dispersions on conventional microscope slides and subsequent Pap staining. A special program was written in RPG II to record and analyze the results of the dissociation experiments in terms of white blood cells and the true cervical cells ("other cells"), and the degree of dissociation and recovery of both classes of cells. Since accurate differential counts on the untreated Ayre scrapes were difficult, the samples were syringed gently to break up the large or adventitious clumps. Cumulated results from control preparations indicate that the white blood cells and "other cells" are composed respectively of 92 and 63% single cells. The cells were further dissociated by: dissolving the cervical mucin sequentially with dithiothreitol and iodoacetic acid; depolymerizing the nucleohistone gel with ribonuclease; solubilizing the desmosomes with EDTA; removing the remaining cellular agglutinins with Varidase; and finally mechanical dispersion by hypertonic shock. The optimum procedure for dissociation involves the use of ribonuclease, dithiothreitol, iodoacetic acid EDTA, Varidase and sucrose shock. The white blood cells are now monodisperse and 81% of the "other cells" are found as single cells. If nuclear separation by two diameters is considered sufficient 98% of the "other cells" are single. The slide preparations are now sufficiently good that a scanning system is feasible.
使用离心细胞学转子在传统显微镜载玻片上制备戊二醛固定的分散液并随后进行巴氏染色，以此监测各种化学和酶处理后宫颈细胞悬液的解离情况。用RPG II编写了一个特殊程序，以便根据白细胞和真正的宫颈细胞（“其他细胞”）以及这两类细胞的解离程度和回收率来记录和分析解离实验的结果。由于对未经处理的艾尔刮片进行准确的差异计数很困难，因此需轻轻用注射器抽吸样本以打散大的或偶然形成的团块。对照制剂的累积结果表明，白细胞和“其他细胞”分别由92%和63%的单细胞组成。通过以下方法进一步解离细胞：依次用二硫苏糖醇和碘乙酸溶解宫颈粘蛋白；用核糖核酸酶使核组蛋白凝胶解聚；用乙二胺四乙酸溶解桥粒；用溶菌酶去除剩余的细胞凝集素；最后通过高渗休克进行机械分散。解离的最佳程序包括使用核糖核酸酶、二硫苏糖醇、碘乙酸、乙二胺四乙酸、溶菌酶和蔗糖休克。现在白细胞呈单分散状态，81%的“其他细胞”为单细胞。如果认为细胞核分离两个直径就足够的话，98%的“其他细胞”是单细胞。现在载玻片制备得足够好，扫描系统是可行的。
