液基薄层制片技术制作外周血淋巴细胞涂片方法学的建立及免疫组化染色应用

Establishment of preparing lymphocytes smears with thinprep liquid-based technology in blood and application of immunostaining

目的应用液基薄层制片技术制作外周血淋巴细胞涂片,以期建立更加简单直观且能够与分子生物学相结合的外周血淋巴细胞形态学检查方法。方法利用Ficoll-hypaque密度梯度离心法分离外周血淋巴细胞,液基细胞保存液中过夜保存,液基薄层涂片技术制作细胞涂片,并通过SP免疫组化染色观察T淋巴细胞及B淋巴细胞表面抗原CD3和CD20在涂片淋巴细胞中的表达,并通过表达部位获取两种抗原的免疫表型保存情况。结果对5名健康者外周静脉血制作淋巴细胞涂片,淋巴细胞获得率达95%以上,形态保存完好,SP免疫组化法显示,CD3和CD20抗原均清晰表达在相应淋巴细胞亚群的细胞膜上,通过免疫组化法计数的淋巴细胞亚群比例和文献结果相一致。结论与传统外周血手工涂片相比,液基薄层制片技术制作外周血淋巴细胞涂片可快速有效地应用于外周血淋巴细胞形态学观察和免疫组化染色,对临床淋巴细胞亚群分类、淋巴瘤分型及预后判断有重要的应用价值。

Objective To develop a new morphological examination method which make smears of lymphocytes in pe-ripheral blood as well as applied with immunohistochemistry using thinprep liquid-based technology. Methods After separated by Ficoll-hypaque density centrifugation, lymphocytes in peripheral blood were preserved in special Preserv-Cyt Solution overnight. And liquid-based smears were prepared with cytospin on the principle of thinprep cytologic test. The immune phenotype of lymphocytes were observed with CD3 and CD20 immunohistochemistry staning. Accu-rate expression location indicated the condition of antigen preservation in lymphocytes. Results The lymphocytes yields of five healthy volunteers reached up to 95% with complete and clear cell morphology. CD3 and CD20 were accord-ingly expressed on cell membranes of two lymphocyte subsets. The proportion of the two lymphocyte subsets calculated by immunohistochemstry staining was in accordance with previous literature. Conclusion The application of preparing lymphocytes smears with thinprep liquid-based technology in blood method in lymphocytes morphology observation and immunohistochemistry staining is rapid and effective compared with conditional manual blood smears. The method also has important values on lymphocyte subsets classification, lymphoma classification and prognosis.