Collste L, Darzynkiewicz Z, Traganos F, Sharpless T K, Whitmore W F, Melamed M R
J Histochem Cytochem. 1979 Jan;27(1):390-3. doi: 10.1177/27.1.86568.
Inflammatory cells are commonly present in cytologic specimens obtained for flow cytometry, and may interfere with the analysis of epithelial cells. We have found that detergent (Triton X-100) pretreatment in the two-step acridine orange staining procedure disrupts granulocyte cell membranes to yield bare nuclei; bladder epithelial and squamous cells on the other hand are quite resistant to the detergent treatment. Being deprived of their cytoplasmic RNA, the granulocytes lose red fluorescence. Moreover, the shearing forces in the cytometer extend the multisegmented granulocyte nuclei and align them in the direction of flow. Thus, they present as elongated objects in the measuring system, giving a large DNA fluorescence pulsewidth (nuclear size). These two phenomena make it possible to identify granulocytes in the recorded data, where they are discernible from the mononucleated leukocytes and from epithelial cells. By data selection the granulocytes can be excluded, rendering epithelial cell populations more amenable to analysis. This method may make it unnecessary to remove physically leukocytes from the specimen before flow cytometry; it may also provide a way to analyze the morphology of granulocyte nuclei and to assess methods to manipulate their membrane stability. Full protection from membrane disruption is accomplished by alcohol fixation, and partial protection by 20-30% serum.
炎症细胞通常存在于用于流式细胞术的细胞学标本中，可能会干扰上皮细胞的分析。我们发现在两步吖啶橙染色程序中用去污剂（曲拉通X-100）预处理会破坏粒细胞细胞膜，产生裸核；另一方面，膀胱上皮细胞和鳞状细胞对去污剂处理具有相当的抗性。粒细胞由于失去了细胞质RNA，失去了红色荧光。此外，细胞仪中的剪切力会使多节段的粒细胞核伸展并使其沿流动方向排列。因此，它们在测量系统中呈现为细长物体，产生较大的DNA荧光脉冲宽度（核大小）。这两种现象使得在记录数据中识别粒细胞成为可能，在这些数据中它们可与单核白细胞和上皮细胞区分开来。通过数据选择可以排除粒细胞，使上皮细胞群体更易于分析。这种方法可能使得在流式细胞术之前无需从标本中物理去除白细胞；它还可能提供一种分析粒细胞核形态和评估操纵其膜稳定性方法的途径。通过酒精固定可实现对膜破坏的完全保护，通过20% - 30%血清可实现部分保护。
