Tests and assays used in the diagnosis of NK cell deficiency

Test	Information provided	Technique	Abnormal result*	Notes
Lymphocyte subset analysis	Identifies and quantifies NK cells	FACS	Absent or decreased	Separate CD56 and CD16 evaluations^¶
NK cytotoxicity and ADCC	Assessment of NK cell function ex vivo	Cytotoxicity assay	Absent or decreased^Δ	Always ship samples with an unrelated control^◊
Cytokine-induced cytotoxicity and cytokine production	Responsiveness to cytokine stimulation (IL-2, IL-12, and IFN-alfa)	Cytotoxicity assay	Absent or decreased
Cytokine-induced cytotoxicity and cytokine production		Intracellular FACS^§	Absent or decreased
NK cell subset analysis	More detailed information about NK cell phenotype	FACS	Missing/decreased activation receptor or increased inhibitory receptor
Redirected lysis and other antibody- or ligand-directed assays	Assessment of specific receptor function	Cytotoxicity assay, granule release, Ca++ flux, and others	Decreased activity after specific receptor ligation

NK: natural killer; FACS: fluorescence-activated cell sorting; ADCC: antibody-dependent cellular cytotoxicity; IL: interleukin; IFN-alfa: interferon-alfa; Ca++: calcium.
* Although it is difficult to interpret a decreased result, values that are persistently lower than the reference range for the laboratory experienced in the investigation may be of significance and worthy of further study.
¶ Many clinical laboratories use a reagent to identify NK cells that contain anti-CD3-FITC as well as both anti-CD56-PE and anti-CD16-PE. Although this reagent will identify NK cells, it will overlook all patients with an alteration of CD16. In suspected cases, both CD16 and CD56 should be evaluated separately. Anti-CD16 clone 3G8 will recognize variant receptors, while clone B73.1 will not. Both reagents are commercially available.
Δ Decreased ex vivo NK cell function in total peripheral blood mononuclear cell (PBMC) preparations may be due to decreased NK cell numbers and thus, may be a defect of NK cell production or survival and not that particular function. If the patient possesses a small population of NK cells, this issue can be addressed using purified or enriched NK cells.
◊ Extreme conditions during shipping can affect biologic activity, and thus, a shipping control is necessary. It is important that the control be unrelated, as some NK deficiencies are present in families.
§ A major cytokine produced by NK cells that is presumably of biologic relevance is IFN-alfa. This factor can be measured intracellularly in NK cells most easily after short-term exposure to mitogen.

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Tests and assays used in the diagnosis of NK cell deficiency