ABSTRACT

Purpose

To develop a more efficient impression cytology (IC) method for the transfer of ocular surface cells onto glass microscope slides for cytochemical, immunocytochemical, and immunofluorescence studies.

Methods

Cells are lifted off the ocular surface with a mixed cellulose ester membrane and then firmly attached to a glass slide using a novel triblock copolymer comprised of collagen type I, polyethylenimine and poly-L-lysine (CPP), and crosslinking cells and glass slide by heating and cooling. The membrane is removed intact after softening it with a butanol/ethanol solution. Transfer of cells is complete in about 10–15 minutes and is ready for staining. The efficiency of our cell transfer method was compared to current methods based on poly-L-lysine and albumin paste.

Results

Our method ensured almost complete transfer of cells. In contrast, the transfer of rabbit conjunctiva cells onto poly-L-lysine-covered slides was 37.5 ± 6.3% lower, and onto albumin-paste covered slides 62.5 ± 5.6% lower (mean ± SD); the transfer of rabbit goblet cells was even less efficient. The new method was also more efficient for transfer of cells from human oral mucosa obtained by IC. Transferred cells were successfully stained with H&E, chemiluminescence, and immunofluorescence agents. Using our method, we stained ocular surface cells for S100A4 and ATF4, both of which play a role in the pathophysiology of dry eye disease. We obtained similar results with oral mucosal cells, suggesting the generalizability of our approach. We propose an explanation for the strong adhesion of cells to the glass slide, which is based on their interactions with the triblock copolymer.

Conclusions

We developed a novel approach for the efficient and rapid transfer of cells obtained by IC onto glass microscope slides using a novel copolymer. Compared to available methods, our improved approach makes IC robust and simple, and should increase its diagnostic yield and clinical applicability.

中文翻译：

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一种改进的眼部印象细胞学方法：使用新型聚合物将细胞定量转移到显微镜载玻片

摘要

目的

开发一种更有效的印象细胞学 (IC) 方法，用于将眼表细胞转移到玻璃显微镜载玻片上，用于细胞化学、免疫细胞化学和免疫荧光研究。

方法

使用混合纤维素酯膜将细胞从眼表面提起，然后使用由 I 型胶原蛋白、聚乙烯亚胺和聚 L-赖氨酸 (CPP) 组成的新型三嵌段共聚物牢固地附着在载玻片上，并交联细胞和载玻片加热和冷却。在用丁醇/乙醇溶液软化后将膜完整地取出。细胞转移在大约 10-15 分钟内完成，可以进行染色。将我们的细胞转移方法的效率与目前基于聚-L-赖氨酸和白蛋白糊的方法进行了比较。

结果

我们的方法确保细胞几乎完全转移。相比之下，兔结膜细胞转移到聚-L-赖氨酸覆盖的载玻片上要低 37.5 ± 6.3%，转移到白蛋白糊覆盖的载玻片上要低 62.5 ± 5.6%（平均值±标准偏差）；兔杯状细胞的转移效率更低。这种新方法对于从 IC 获得的人口腔粘膜细胞转移也更有效。转移的细胞成功地用 H&E、化学发光和免疫荧光剂染色。使用我们的方法，我们对 S100A4 和 ATF4 的眼表细胞进行了染色，这两者在干眼病的病理生理学中都发挥着重要作用。我们用口腔粘膜细胞获得了类似的结果，表明我们的方法具有普遍性。我们提出了细胞对载玻片的强粘附性的解释，

结论

我们开发了一种新方法，用于使用新型共聚物将 IC 获得的细胞高效快速地转移到玻璃显微镜载玻片上。与现有方法相比，我们改进的方法使 IC 稳健且简单，并且应该提高其诊断率和临床适用性。