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Updated: Dec 9, 2025
Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood
Published on: March 17, 2017
A Flow Cytometry-Based Whole Blood Natural Killer Cell Cytotoxicity Assay Using Overnight Cytokine Activation
Jinho Kim1, Minh-Trang Thi Phan2, SoonHo Kweon2,3
1Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul, South Korea.
Insights
A new overnight whole blood assay accurately measures natural killer (NK) cell function, correlating well with existing methods. This assay shows reduced NK cell activity in liver disease patients, offering potential for clinical research.
Area of Science:
- Immunology
- Clinical Laboratory Science
- Hepatology
Background:
- Natural killer (NK) cell function measurement is clinically relevant for various diseases.
- Conventional flow cytometry (FC)-based 4-hour NK cytotoxicity assays using peripheral blood mononuclear cells (PBMCs) require large blood volumes and rapid PBMC isolation.
- A simplified, effective NK cell function assay is needed for clinical utility.
Purpose of the Study:
- To develop and validate an FC-based overnight NK cytotoxicity assay using whole blood (WB).
- To assess NK cell activity in patients with liver diseases using the novel assay.
- To compare the novel WB assay with the conventional PBMC-based assay.
Main Methods:
- Developed an FC-based overnight NK cytotoxicity assay using WB and K562 target cells stimulated with cytokine combinations (IL-2, IL-18, IL-21).
- Analyzed peripheral blood from healthy volunteers (n=28) and liver disease patients (hepatocellular carcinoma, n=19; liver cirrhosis, n=7).
- Compared the overnight WB assay with a 4-hour PBMC assay and an FC-based CD107a degranulation assay.
Main Results:
- Optimal cytokine combinations for NK cell activation in WB were identified (IL-2/IL-18, IL-2/IL-21, IL-2/IL-18/IL-21).
- The overnight WB NK cytotoxicity assay showed significant correlation with the 4-hour PBMC assay.
- Patients with liver diseases exhibited significantly lower NK cytotoxicity compared to healthy controls (p < 0.001).
Conclusions:
- The FC-based overnight WB NK cytotoxicity assay is a reliable alternative to the conventional PBMC assay.
- This assay demonstrates reduced NK cell function in patients with liver diseases.
- The assay holds potential for clinical laboratory research and observational studies, especially in liver disease.
Abstract:
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.
