Th细胞亚群测定中通透剂作用效果的评价

Evaluation of the effect of permeabilization in measuring Th subsets

目的 建立一种在Th细胞亚群测定中评价通透剂作用效果的方法。方法 将刺激培养并固定后的全血标本在通透剂作用前及用不同剂量的通透剂作用后 ,加入荧光素标记的CD 6 9抗体、髓过氧化物酶 (MPO)抗体和γ干扰素(IFN γ)抗体 ,用流式细胞仪检测含相应抗原的细胞。结果 通透剂作用前及不同剂量的通透剂作用后 ,T淋巴细胞中CD6 9+细胞的百分率均大于 90 % ;通透剂作用前 ,MPO阳性的粒细胞和IFN γ阳性的淋巴细胞均低于 1% ;使用 30 μl通透剂时 ,粒细胞几乎全部为MPO阳性 ,>30 %的淋巴细胞IFN γ阳性 ;使用 5 0 μl通透剂时 ,总细胞数比通透剂作用前显著减少 (t =13 6 2 3,P <0 0 0 1)。结论 MPO可以作为评价通透剂作用效果的指标 ;当细胞数同通透剂作用前无显著差异 ,且粒细胞门的细胞几乎全部被染为MPO阳性时 ,所用的通透剂量为最适用量。

Objective To establish a method for evaluating the effects of permeabilization in measuring Th subsets.Methods The whole blood samples were co-cultured with stimulis and fixed,then fluorescein-labeled antibody of CD69, myeloperoxidase(MPO)and interferon-γ(IFN-γ) were added before adding permeabilization medium and after adding different dosages of permeabilization medium. The cells containing corresponding antigen were measured by flow cytometry.Results Before adding permeabilization medium and after adding different dosages of permeabilization medium, the percentage of CD3+ cells expressing CD69 exceeded 90%. Before adding permeabilization medium, the percentages of leukocytes of MPO+ and lym-phocytes of IFN-γ+ were both lower than 1%. When adding 30 μl of permeabilization medium, almost all of the leukocytes were MPO+,and the percentage of lymphocytes of IFN-γ+ exceeded 30%. When adding 50 μl of permeabilization medium, the cell counts were significant lower than that before adding permeabilization medium.Conclusion MPO could be the marker for evaluating the effects of permeabilization. When the cell counts were not significant different before and after using permeabilization and almost all of the leukocytes were MPO+, the dosage of permeabilization was the optimal.