J Lab Med Qual Assur.  2012 Jun;34(1):1-8.

CD45 is Essential for Lymphocyte Gating in a T-lymphocyte Subset Assay of Bronchoalveolar Lavage Fluid by Flow Cytometry

• Shim H
• Choi YH
• Park CJ
• Lee JS
• Han SH
• Hwang K
• Jang S
• Chi HS

Abstract

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.

Keyword

Bronchoalveolar lavage fluid; Flow cytometry; Lymphocyte gating; T-lymphocytes subsets

MeSH Terms

Bronchoalveolar Lavage
Bronchoalveolar Lavage Fluid
Flow Cytometry
Hematology
Leukocyte Count
Light
Lymphocyte Subsets
Lymphocytes
T-Lymphocyte Subsets
T-Lymphocytes