Lab Med Online.  2013 Apr;3(2):79-87.

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

Affiliations
  • 1Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea. cjpark@amc.seoul.kr

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.

Keyword

Flow cytometry; Lymphocyte Gating; CD45; NK cells; CD16
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