A Flow Cytometry-Based Whole Blood Natural Killer Cell Cytotoxicity Assay Using Overnight Cytokine Activation

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.

An automated real-time microfluidic platform to probe single NK cell heterogeneity and cytotoxicity on-chip

“…Bulk-based studies fail to address the functional heterogeneity underlying a given cell population by masking the phenotype, gene expression, and the mechanism of cellular communication in between individual immune cells 11 , 12 . To overcome the limitations of bulk methodologies, the study of cytotoxicity at a single-cell level can be performed using flow cytometry-based assays and single cell microscopy assays 13 , 14 . However, flow cytometric assays are snapshot-based and therefore not suited to monitor temporal dynamics of cellular interactions leading to cytotoxicity 10 .…”

Lessons learned from immunological characterization of nanomaterials at the Nanotechnology Characterization Laboratory

“…Other experimental approaches include whole-blood and PBMC cytotoxicity assays in which CFSE-labeled K562 target cells are monitored by flow cytometry to assess the cytotoxicity of primary effector NK cells. Another flow-cytometry-based approach includes the CD107a degranulation assay, in which whole blood or PBMCs serve as the source of primary NK cells ( 225 , 226 ).…”

The Role of NK Cells in Recurrent Miscarriage (Abortion)

“…It binds ligands induced in stressed cells and therefore alerting immune cells' function. While these ligands are not found on trophoblast, they can be induced on T-cell immunoglobulin domain and mucin domain-containing molecule-3 (Tim-3) is a newly defined regulatory factor that can modulate the balance of Th1 cells/Th2 cells and expresses in some dNK cells, and Tim-3+ dNK+ cells displayed decreased cytotoxicity due to producing less perforin [15,16].…”