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Updated: Dec 19, 2025
Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood
Published on: March 17, 2017
A Method of Assessment of Human Natural Killer Cell Phenotype and Function in Whole Blood
Marisa Market1, Gayashan Tennakoon2, Juliana Ng2
1University of Ottawa, Ottawa, ON, Canada.
Insights
This study presents a novel whole blood method for analyzing human Natural Killer (NK) cell function, bypassing cryopreservation artifacts. The approach allows for rapid, parallel assessment of NK cell receptors, signaling, and cytokine production in minimal blood volumes from healthy donors and cancer patients.
Area of Science:
- Immunology
- Cell Biology
- Cancer Research
Background:
- Cryopreservation of peripheral blood mononuclear cells (PBMCs) for human Natural Killer (NK) cell analysis can introduce artifacts and requires significant sample volumes.
- Investigating immune cells directly in whole blood offers a more time-efficient and less artifact-prone alternative.
Purpose of the Study:
- To develop and validate protocols for assessing human NK cell phenotype and function using whole blood.
- To enable parallel analysis of extracellular receptors, intracellular signaling, and cytokine production in NK cells.
- To compare NK cell marker expression in whole blood versus cryopreserved PBMCs.
Main Methods:
- Three distinct protocols were applied to whole blood from healthy donors and cancer patients to analyze extracellular receptors, intracellular signaling protein phosphorylation, and cytokine production.
- Methods involved specific incubation steps with stimulation agents (e.g., IL-2/12, PMA-ionomycin), extracellular staining (ECS), intracellular staining (ICS), cell fixation, and red blood cell (RBC) lysis.
- Quantification of secreted IFNγ was performed using ELISA from plasma samples.
Main Results:
- The developed whole blood methods consistently quantified extracellular receptors (e.g., NKG2D, PD-1), intracellular signaling markers (e.g., p-STAT4, p-STAT5), and cytokine production (IFNγ) in NK cells.
- Significant differences in extracellular receptor expression were observed when comparing whole blood analysis to cryopreserved PBMCs.
- The approach demonstrated effectiveness across healthy donors and cancer patients, despite inter-patient variability.
Conclusions:
- Whole blood analysis provides a rapid and efficient method for assessing human NK cell receptor expression, signaling activity, and cytokine production.
- This method minimizes artifacts associated with cryopreservation and requires a smaller blood volume.
- The validated protocols offer a valuable tool for immunological studies in both healthy individuals and cancer patients.
Abstract:
The majority of data on human Natural Killer (NK) cell phenotype and function has been generated using cryopreserved peripheral blood mononuclear cells (PBMCs). However, cryopreservation can have adverse effects on PBMCs. In contrast, investigating immune cells in whole blood can reduce the time, volume of blood required, and potential artefacts associated with manipulation of the cells. Whole blood collected from healthy donors and cancer patients was processed by three separate protocols that can be used independently or in parallel to assess extracellular receptors, intracellular signaling protein phosphorylation, and intracellular and extracellular cytokine production in human NK cells. To assess extracellular receptor expression, 200 μL of whole blood was incubated with an extracellular staining (ECS) mix and cells were subsequently fixed and RBCs lysed prior to analysis. The phosphorylation status of signaling proteins was assessed in 500 μL of whole blood following co-incubation with interleukin (IL)-2/12 and an ECS mix for 20 min prior to cell fixation, RBC lysis, and subsequent permeabilization for staining with an intracellular staining (ICS) mix. Cytokine production (IFNγ) was similarly assessed by incubating 1 mL of whole blood with PMA-ionomycin or IL-2/12 prior to incubation with ECS and subsequent ICS antibodies. In addition, plasma was collected from stimulated samples prior to ECS for quantification of secreted IFNγ by ELISA. Results were consistent, despite inherent inter-patient variability. Although we did not investigate an exhaustive list of targets, this approach enabled quantification of representative ECS surface markers including activating (NKG2D and DNAM-1) and inhibitory (NKG2A, PD-1, TIGIT, and TIM-3) receptors, cytokine receptors (CD25, CD122, CD132, and CD212) and ICS markers associated with NK cell activation following stimulation, including signaling protein phosphorylation (p-STAT4, p-STAT5, p-p38 MAPK, p-S6) and IFNγ in both healthy donors and cancer patients. In addition, we compared extracellular receptor expression using whole blood vs. cryopreserved PBMCs and observed a significant difference in the expression of almost all receptors. The methods presented permit a relatively rapid parallel assessment of immune cell receptor expression, signaling protein activity, and cytokine production in a minimal volume of whole blood from both healthy donors and cancer patients.
