Prince H E, York J, Kuttner D K
American Red Cross Blood Services, Southern California Region, Los Angeles.
J Immunol Methods. 1994 Dec 28;177(1-2):165-73. doi: 10.1016/0022-1759(94)90153-8.
Most experienced flow cytometrists performing immunophenotyping of lymphocyte subsets are aware of the escape phenomenon, in which some positive cells are found outside a lymphocyte gate based on forward and right angle scatter. However, little information is available on the levels of escapees formed with different antibodies, the roles of fluorochromes and lysing agents, the mechanism explaining the phenomenon, or methods to reduce it. We thus performed a systematic analysis of the escapee phenomenon to clarify these issues. A panel of monoclonal antibodies, including a phycoerythrin (PE) conjugate and a fluorescein isothiocyanate (FITC) conjugate of the same antibody from one manufacturer, was used to treat whole blood specimens, after which red cells were lysed using 0.15 M ammonium chloride (AmChl). The percentages of gated lymphocytes expressing CD3, CD8, CD19, and HLA-DR, but not CD2, CD4, CD16, and CD25, were significantly lower in FITC-stained versus PE-stained preparations. Correlated analysis of green fluorescence and forward scatter showed that, on average, 18% of CD3+ events, 24% of CD8+ events, and 25% of CD19+ events were escapees when using the FITC conjugate. In dual color analysis, CD3+ escapees were positive for CD62-P, CD13, and CD14, indicating that the escapee events consisted of FITC-anti-CD3-coated lymphocytes complexed with platelet-coated myeloid cells. In studies of the role of lysing agent, essentially no escapees were found in specimens treated with FACS lysing solution, which contains formaldehyde. We therefore included a similar denaturing agent, paraformaldehyde (0.1%), in the AmChl lysing agent, and found that the occurrence of escapees was markedly reduced. These findings show that the escapee phenomenon occurs when using some FITC-conjugated monoclonal antibodies in conjunction with AmChl lysing agent, and can be reduced by inclusion of paraformaldehyde in the lysing agent.
大多数进行淋巴细胞亚群免疫表型分析的经验丰富的流式细胞仪操作人员都知道逃逸现象，即在基于前向散射和直角散射的淋巴细胞门之外发现一些阳性细胞。然而，关于不同抗体形成的逃逸细胞水平、荧光染料和裂解剂的作用、解释该现象的机制或减少它的方法，几乎没有可用信息。因此，我们对逃逸现象进行了系统分析以阐明这些问题。使用一组单克隆抗体，包括来自同一制造商的同一抗体的藻红蛋白（PE）偶联物和异硫氰酸荧光素（FITC）偶联物，处理全血标本，然后用0.15M氯化铵（AmChl）裂解红细胞。在FITC染色的制剂中，表达CD3、CD8、CD19和HLA-DR但不表达CD2、CD4、CD16和CD25的门控淋巴细胞百分比显著低于PE染色的制剂。绿色荧光与前向散射的相关分析表明，使用FITC偶联物时，平均18%的CD3+事件、24%的CD8+事件和25%的CD19+事件是逃逸细胞。在双色分析中，CD3+逃逸细胞对CD62-P、CD13和CD14呈阳性，表明逃逸事件由与血小板包被的髓样细胞复合的FITC-抗CD3包被的淋巴细胞组成。在裂解剂作用的研究中，在用含有甲醛的FACS裂解液处理的标本中基本未发现逃逸细胞。因此，我们在AmChl裂解剂中加入了一种类似的变性剂多聚甲醛（0.1%），发现逃逸细胞的发生率显著降低。这些发现表明，在使用一些FITC偶联的单克隆抗体与AmChl裂解剂结合时会出现逃逸现象，并且通过在裂解剂中加入多聚甲醛可以减少这种现象。
