A Flow Cytometry-Based Whole Blood Natural Killer Cell Cytotoxicity Assay Using Overnight Cytokine Activation
Background: Measurement of natural killer (NK) cell function has important clinical utility in several diseases. Although the flow cytometry (FC)-based 4-h NK cytotoxicity assay using peripheral blood mononuclear cells (PBMCs) in the clinical laboratory has been used for this purpose, this assay req...
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doaj-ed9a150f52e040a9877f273015c078852020-11-25T03:53:13ZengFrontiers Media S.A.Frontiers in Immunology1664-32242020-08-011110.3389/fimmu.2020.01851519027A Flow Cytometry-Based Whole Blood Natural Killer Cell Cytotoxicity Assay Using Overnight Cytokine ActivationJinho Kim0Minh-Trang Thi Phan1SoonHo Kweon2SoonHo Kweon3HongBi Yu4Jeehun Park5Kyeong-Hee Kim6Ilwoong Hwang7Sangbin Han8Min-Jung Kwon9Duck Cho10Duck Cho11Duck Cho12Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul, South KoreaSamsung Medical Center, Stem Cell & Regenerative Medicine Institute, Seoul, South KoreaSamsung Medical Center, Stem Cell & Regenerative Medicine Institute, Seoul, South KoreaResearch Institute of Advanced Materials, Seoul National University, Seoul, South KoreaDepartment of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul, South KoreaResearch Institute of Advanced Materials, Seoul National University, Seoul, South KoreaDepartment of Laboratory Medicine, Dong-A University College of Medicine, Busan, South KoreaDepartment of Emergency Medicine, Konkuk University Chungju Hospital, Chungju, South KoreaDepartment of Anesthesiology and Pain Medicine Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South KoreaDepartment of Laboratory Medicine, Kangbuk Samsung Hospital, Sungkyunkwan University School of Medicine, Seoul, South KoreaDepartment of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul, South KoreaSamsung Medical Center, Stem Cell & Regenerative Medicine Institute, Seoul, South KoreaDepartment of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South KoreaBackground: Measurement of natural killer (NK) cell function has important clinical utility in several diseases. Although the flow cytometry (FC)-based 4-h NK cytotoxicity assay using peripheral blood mononuclear cells (PBMCs) in the clinical laboratory has been used for this purpose, this assay requires large amounts of blood and a rapid PBMC isolation step. Here, we developed an FC-based overnight NK cytotoxicity assay using whole blood (WB), and applied it to patients with liver diseases.Methods: Peripheral blood of healthy volunteers (n = 28) and patients with liver diseases, including hepatocellular carcinoma (n = 19) and liver cirrhosis (n = 7), was analyzed for complete blood count, absolute NK cell count, and NK cell activity (NKA). NKA was evaluated in three assay types: an FC-based overnight WB NK cytotoxicity assay using carboxyfluorescein diacetate succinimidyl ester-labeled K562 cells in the presence of various cytokine combinations [including interleukin (IL)-2, IL-18, and IL-21], an FC-based 4-h PBMC NK cytotoxicity assay, and an FC-based CD107a degranulation assay using WB and PBMCs.Results: Optimal cytokine combinations for NK cell activation in WB were determined (IL-2/IL-18, IL-2/IL-21, and IL-2/IL-18/IL-21). A good correlation was observed between WB and PBMC NK cytotoxicity assays; absolute NK cell counts were better correlated with the WB NK cytotoxicity assay than with the PBMC NK cytotoxicity assay. This WB NK cytotoxicity assay showed that patients with liver diseases had significantly lower NK cytotoxicity than healthy volunteers, under stimulation with various cytokines (p < 0.001).Conclusion: The proposed FC-based overnight WB NK cytotoxicity assay correlates well with the conventional 4-h PBMC NK cytotoxicity assay, demonstrating future potential as a supportive assay for clinical laboratory research and observational studies.https://www.frontiersin.org/article/10.3389/fimmu.2020.01851/fullnatural killer cellsactivity assaycytotoxicitywhole bloodcytokines |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Jinho Kim Minh-Trang Thi Phan SoonHo Kweon SoonHo Kweon HongBi Yu Jeehun Park Kyeong-Hee Kim Ilwoong Hwang Sangbin Han Min-Jung Kwon Duck Cho Duck Cho Duck Cho |
spellingShingle |
Jinho Kim Minh-Trang Thi Phan SoonHo Kweon SoonHo Kweon HongBi Yu Jeehun Park Kyeong-Hee Kim Ilwoong Hwang Sangbin Han Min-Jung Kwon Duck Cho Duck Cho Duck Cho A Flow Cytometry-Based Whole Blood Natural Killer Cell Cytotoxicity Assay Using Overnight Cytokine Activation Frontiers in Immunology natural killer cells activity assay cytotoxicity whole blood cytokines |
author_facet |
Jinho Kim Minh-Trang Thi Phan SoonHo Kweon SoonHo Kweon HongBi Yu Jeehun Park Kyeong-Hee Kim Ilwoong Hwang Sangbin Han Min-Jung Kwon Duck Cho Duck Cho Duck Cho |
author_sort |
Jinho Kim |
title |
A Flow Cytometry-Based Whole Blood Natural Killer Cell Cytotoxicity Assay Using Overnight Cytokine Activation |
title_short |
A Flow Cytometry-Based Whole Blood Natural Killer Cell Cytotoxicity Assay Using Overnight Cytokine Activation |
title_full |
A Flow Cytometry-Based Whole Blood Natural Killer Cell Cytotoxicity Assay Using Overnight Cytokine Activation |
title_fullStr |
A Flow Cytometry-Based Whole Blood Natural Killer Cell Cytotoxicity Assay Using Overnight Cytokine Activation |
title_full_unstemmed |
A Flow Cytometry-Based Whole Blood Natural Killer Cell Cytotoxicity Assay Using Overnight Cytokine Activation |
title_sort |
flow cytometry-based whole blood natural killer cell cytotoxicity assay using overnight cytokine activation |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Immunology |
issn |
1664-3224 |
publishDate |
2020-08-01 |
description |
Background: Measurement of natural killer (NK) cell function has important clinical utility in several diseases. Although the flow cytometry (FC)-based 4-h NK cytotoxicity assay using peripheral blood mononuclear cells (PBMCs) in the clinical laboratory has been used for this purpose, this assay requires large amounts of blood and a rapid PBMC isolation step. Here, we developed an FC-based overnight NK cytotoxicity assay using whole blood (WB), and applied it to patients with liver diseases.Methods: Peripheral blood of healthy volunteers (n = 28) and patients with liver diseases, including hepatocellular carcinoma (n = 19) and liver cirrhosis (n = 7), was analyzed for complete blood count, absolute NK cell count, and NK cell activity (NKA). NKA was evaluated in three assay types: an FC-based overnight WB NK cytotoxicity assay using carboxyfluorescein diacetate succinimidyl ester-labeled K562 cells in the presence of various cytokine combinations [including interleukin (IL)-2, IL-18, and IL-21], an FC-based 4-h PBMC NK cytotoxicity assay, and an FC-based CD107a degranulation assay using WB and PBMCs.Results: Optimal cytokine combinations for NK cell activation in WB were determined (IL-2/IL-18, IL-2/IL-21, and IL-2/IL-18/IL-21). A good correlation was observed between WB and PBMC NK cytotoxicity assays; absolute NK cell counts were better correlated with the WB NK cytotoxicity assay than with the PBMC NK cytotoxicity assay. This WB NK cytotoxicity assay showed that patients with liver diseases had significantly lower NK cytotoxicity than healthy volunteers, under stimulation with various cytokines (p < 0.001).Conclusion: The proposed FC-based overnight WB NK cytotoxicity assay correlates well with the conventional 4-h PBMC NK cytotoxicity assay, demonstrating future potential as a supportive assay for clinical laboratory research and observational studies. |
topic |
natural killer cells activity assay cytotoxicity whole blood cytokines |
url |
https://www.frontiersin.org/article/10.3389/fimmu.2020.01851/full |
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