Pizzolo J G, Melamed M R
Department of Pathology, Flow Cytometry Lab, New York Medical College, Elmsford, New York, USA.
Cytometry. 1996 Jan 1;23(1):67-71. doi: 10.1002/(SICI)1097-0320(19960101)23:1<67::AID-CYTO10>3.0.CO;2-U.
Third-color CD45 gating is a useful procedure in various clinical applications (Borowitz et al.: Am J Clin Pathol 100:534-540, 1993; Steltzer et al.: Ann NY Acad Sci 677:267-280, 1993) allowing for the selective immunophenotyping of abnormal cell populations. In laboratories where dual-color protocols are in place, the need for CD45 gating may not become evident until after cells have been exposed to fixatives. This brief study demonstrates that third-color CD45 staining and gating is possible with paraformaldehyde fixed lymphocytes, eliminating the need to restain fresh cells with the entire antibody panel, and with a significant saving of time and money. Gates must be modified from those obtained by fresh cell staining in order to include both bright and dim CD45 populations. In a standard protocol, third-color CD45 staining intensity was decreased 10-fold post-fixation with paraformaldehyde except, unexpectedly, for CD8+ cells. The latter phenomenon could be explained if the spatial arrangement of CD45 and CD8 is such that the CD45 epitope is protected from cross-linking by paraformaldehyde fixation in CD8+ cells, but not others.
三色CD45设门在各种临床应用中是一种有用的方法（博罗维茨等人：《美国临床病理学杂志》100:534 - 540，1993年；施特尔策等人：《纽约科学院学报》677:267 - 280，1993年），可用于异常细胞群体的选择性免疫表型分析。在采用双色方案的实验室中，直到细胞接触固定剂后，CD45设门的必要性才可能显现出来。这项简短的研究表明，用多聚甲醛固定的淋巴细胞进行三色CD45染色和设门是可行的，无需用完整的抗体组合对新鲜细胞重新染色，从而显著节省了时间和金钱。必须对通过新鲜细胞染色获得的门进行修改，以纳入明亮和暗淡的CD45群体。在标准方案中，用多聚甲醛固定后，三色CD45染色强度降低了10倍，但出乎意料的是，CD8 +细胞除外。如果CD45和CD8的空间排列使得CD45表位在CD8 +细胞中受到保护，免受多聚甲醛固定的交联影响，而在其他细胞中则不然，那么后一种现象就可以得到解释。
