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J Lab Med Qual Assur. 2012 Jun;34(1):1-8. English. Original Article.
Shim H , Choi YH , Park CJ , Lee JS , Han SH , Hwang K , Jang S , Chi HS .
Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea.

BACKGROUND: Proper gating is important in flow cytometric assays of lymphocyte subsets. Forward light scatter (FSC)/side light scatter (SSC) gating requires application of a lymphocyte purity correction when lymphocyte purity is less than 95%. We compared 3 different gating methods to establish an accurate gating method appropriate for a T-lymphocyte subset assay of bronchoalveolar lavage (BAL) fluid. METHODS: Leukocyte numbers and subtypes in 31 BAL fluid samples were assessed manually and by using an automatic hematology analyzer. T-lymphocyte subsets (T cells, T helper/inducer cells [Th], and T suppressor/cytotoxic cells [Tc]) were assessed by flow cytometry. We compared 3 methods of lymphocyte gating: CD45/SSC gating (reference method), FSC/SSC gating, and FSC/SSC gating with application of a lymphocyte purity correction. Lymphocyte purity was determined by CD45/CD14 staining of BAL fluid. RESULTS: We observed a significant correlation between lymphocyte percentage and lymphocyte purity (r = 0.453, P = 0.011). T-cell results obtained using the reference method were not correlated with the results of the other 2 gating methods (r = 0.189 each, P = 0.308 for FSC/SSC gating and P = 0.310 for FSC/SSC gating with purity correction). Mean differences between the reference method and FSC/SSC gating (T cells: 14.4%, P = 0.002; Th cells: 7.7%, P = 0.006; Tc cells: 7.1%, P = 0.001) were greater than those between the reference method and FSC/SSC gating with purity correction (T cells: 12.1%, P = 0.004; Th cells: 1.7%, P = 0.608; Tc cells: 0.2%, P = 0.957). CONCLUSIONS: Lymphocyte purity correction after FSC/SSC gating improved the accuracy of Th- and Tc-cell measurements, but not T-cell measurements. CD45 is essential for lymphocyte gating in T-lymphocyte subset assays of BAL fluid.

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