Albro J, Bauer K D, Hitchcock C L, Wittwer C T
Department of Pathology, University of Utah Medical School, Salt Lake City 84132.
Cytometry. 1993;14(6):673-8. doi: 10.1002/cyto.990140612.
An improved method for the enzymatic digestion of formalin-fixed, paraffin-embedded liver tissue for DNA content analysis by flow cytometry is presented. Forty samples of histologically normal liver were alternately digested by the traditional pepsin method or a new method utilizing proteinase K and heat. Sixteen (40%) of the pepsin-digested samples had apparent DNA aneuploid peaks by flow cytometry. False DNA aneuploid peaks were not present in any of the histograms obtained after proteinase K digestion. Microscopy showed that the pepsin-digested samples had residual cytoplasmic remnants which contained fluorescent material. Samples digested with proteinase K had few cytoplasmic remnants. The average G0/G1 coefficient of variation after proteinase K treatment was lower (41%) and the fluorescent intensity higher (128%) than the pepsin-treated samples. The apparent mean S-phase (a combination of S-phase cells and underlying debris) after proteinase K digestion was 35% of the pepsin-treated samples. Primary and secondary tumors of the liver that were DNA aneuploid after pepsin treatment were also DNA aneuploid after proteinase K treatment. A modified digestion protocol utilizing proteinase K and heat can provide superior results for DNA content analysis of formalin-fixed, paraffin-embedded liver tissue.
本文介绍了一种改进的酶消化方法，用于对福尔马林固定、石蜡包埋的肝组织进行酶消化，以通过流式细胞术分析DNA含量。选取40份组织学正常的肝脏样本，分别采用传统的胃蛋白酶法或利用蛋白酶K和加热的新方法进行交替消化。经胃蛋白酶消化的样本中，有16份（40%）通过流式细胞术检测到明显的DNA非整倍体峰。蛋白酶K消化后得到的任何直方图中均未出现假DNA非整倍体峰。显微镜检查显示，经胃蛋白酶消化的样本有残留的细胞质残余物，其中含有荧光物质。用蛋白酶K消化的样本细胞质残余物较少。蛋白酶K处理后的平均G0/G1变异系数较低（41%），荧光强度较高（128%），均高于经胃蛋白酶处理的样本。蛋白酶K消化后的表观平均S期（S期细胞与下层碎片的组合）为经胃蛋白酶处理样本的35%。经胃蛋白酶处理后为DNA非整倍体的原发性和继发性肝肿瘤，经蛋白酶K处理后也为DNA非整倍体。利用蛋白酶K和加热的改良消化方案可为福尔马林固定、石蜡包埋的肝组织的DNA含量分析提供更好的结果。
