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

Young-Hyun Choi; Hyoeun Shim; Chan-Jeoung Park; Sang Hee Han; Keumrock Hwang; Seongsoo Jang; Hyun-Sook Chi

doi:10.3343/lmo.2013.3.2.79

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Choi, Shim, Park, Han, Hwang, Jang, and Chi: Flow Cytometric Assays for Lymphocyte Subset Enumeration: CD45 is Inevitable for Lymphocyte Gating and CD16 is Essential for NK Cells

Original Article

Diagnostic Immunology

Laboratory Medicine Online

Laboratory Medicine Online 2013; 3(2): 79-87.

Published online: 1 April 2013

DOI: https://doi.org/10.3343/lmo.2013.3.2.79

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

Young-Hyun Choi, M.D., Hyoeun Shim, M.D., Chan-Jeoung Park, M.D., Sang Hee Han, M.T., Keumrock Hwang, M.D., Seongsoo Jang, M.D., Hyun-Sook Chi, M.D.

Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea.

Corresponding author: Chan-Jeoung Park, MD. Department of Laboratory Medicine, Asan Medical Center, 86 Asanbyeongwon-gil, Songpa-gu, Seoul 138-736, Korea. Tel: +82-2-3010-4508, Fax: +82-2-478-0884, cjpark@amc.seoul.kr

Received 18 March 2012 Revised 24 August 2012 Accepted 27 August 2012

(open-access):

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Keywords: Flow cytometry, Lymphocyte Gating, CD45, NK cells, CD16

Figures and Tables

Fig. 1

Assay for enumerating the lymphocyte subset using 3 different gating methods. (A) CD45/SSC gating. (B) FSC/SSC gating. (C) Gate purity correction after FSC/SSC gating. The corrected lymphocyte subset (%) was calculated using the following formulae: ^*Gate purity (%)=CD45(+) bright/CD14(-) mononuclear cells (%) among lymphocytes by using FSC/SSC gating.

Corrected lymphocyte subset result (%) = \frac{Lymphocyte subset result from FSC/SSC gating (%)}{Gate purity (%)} \times 100

Fig. 2

Evaluation of NK cell subset by using CD16 and/or CD56 expression.

Fig. 3

(A) Correlation between the proportion of lymphocytes and lymphocyte gate purities (r=0.400, P<0.001). (B) Correlation between the proportion of lymphocytes and the differences in T cell values analyzed using CD45/SSC gating and lymphocyte gate purity correction after FSC/SSC gating (r=-0.133, P=0.115).

Fig. 4

Distribution of NK cell subsets in patients with various diseases.

^*Proportion of NK cells that are CD56-CD16+ NK cells.

Abbreviations: NC, normal control; AML, follow-up of patients with acute myeloid leukemia after chemotherapy; ALL, follow-up of patients with acute lymphoblastic leukemia after chemotherapy; MDS, myelodysplastic syndrome; LC, liver cirrhosis; AA, aplastic anemia; VI, viral infection.

Table 1

Percentages of lymphocytic subsets calculated using 3 different lymphocyte gating methods (N=114)

^*All, P<0.001.

A, CD45/SSC gating; B, FSC/SSC gating; C, Lymphocyte gate purity correction after FSC/SSC gating

Table 2

Distribution of NK cell subsets according to disease entity

^*P<0.05, when data of the NK subset from patient groups were compared with those of the normal controls by using the Mann-Whitney test.

Abbreviations: NC, normal control; VI, viral infection; AML, follow-up of patients with acute myeloid leukemia after chemotherapy; ALL, follow-up of acute lymphoblastic leukemia after chemotherapy; MDS, myelodysplastic syndrome; LC, liver cirrhosis; AA, aplastic anemia.

Notes

This article is available from http://www.labmedonline.org

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