Flow Cytometric Assays for Lymphocyte Subset Enumeration: CD45 is Inevitable for Lymphocyte Gating and CD16 is Essential for NK Cells

ABSTRACT

BACKGROUND: Clinical and Laboratory Standards Institute (CLSI) guidelines (H42-A2) recommend the "CD45/SSC" gating method for assays on lymphocyte subset enumeration and CD16 exclusion for assays enumerating NK cells. In contrast, the Flow Cytometry Checklist (06/17/2010) of the College of American Pathology does not recommend a specific lymphocyte gating method, but recommends the correction of lymphocyte subset results for lymphocyte gate purity. METHODS: We compared lymphocyte subset results of EDTA-treated blood from 102 patients with various diseases and 12 normal controls, using 3 lymphocyte gating methods (CD45/SSC, FSC/SSC, and lymphocyte gate purity correction after FSC/SSC gating), and assessed the proportion of CD56-/CD16+ NK cells within the total NK cell population. RESULTS: Lymphocyte gate purity increased as the percentage of lymphocytes increased. However, lymphocyte subsets that consistently showed high lymphocyte gate purity could not be identified. The purity of the T cell population differed significantly depending on the gating method used CD45/SSC vs. FSC/SSC, P=0.027; CD45/SSC vs. gate purity correction after FSC/SSC, P=0.002. However, the lymphocyte gate purity correction after FSC/SSC gating did not significantly improve the accuracy of the lymphocyte subset enumeration assay using FSC/SSC gating. The subset of CD56-CD16+ NK cells, constituted an average of 17.1% of total NK cells. Patients had higher proportions of CD56-CD16+ NK cells (13.1-25.5%) than did the normal controls (9.52%). CONCLUSIONS: In flow cytometric assays to evaluate lymphocytic subsets, the CD45 is inevitable for lymphocyte gating, whereas the measurement of CD16 is essential for the evaluation of NK cell proportions.