Test Id : QNKS

This assay does not measure cell-surface or intracellular proteins on natural killer or natural killer T-cell subsets.

A minimum CD45 lymph count (as measured by flow cytometry in the laboratory) is required to report this test. If that requirement is not met (eg, patients with severe lymphopenia), the test will be canceled and an alternate test will be suggested (TBBS / Quantitative Lymphocyte Subsets: T, B, and Natural Killer [NK] Cells, Blood).

Send specimen Monday through Thursday only. Specimen must arrive within 24 hours of collection and by 10 a.m. on Friday. Collect and package specimen as close to shipping time as possible. Ship specimen overnight.

Ordering physician's name and phone number are required.

Container/Tube: Green top (sodium heparin)

Specimen Volume: 3 mL

Collection Instructions: Send whole blood specimen in original tube. Do not aliquot.

Additional Information: For serial monitoring, it is recommended that specimens are collected at the same time of day.

0.2 mL

Quantitation of the major natural killer (NK)-cell subsets relative to total NK cells (NK cell subsets) or total lymphocytes (NK T cells)

Assessment in the following clinical contexts: HIV, primary immune deficiencies with NK cell defects, NK-cell lymphocytosis, solid-organ transplantation, immune reconstitution following bone marrow or hematopoietic cell transplantation

This test is not useful for diagnosis or classification of NK cell malignancies.

This test should not be used for assessing NK cell cytotoxic function.

Natural killer (NK) cells are derived from pluripotent hematopoietic stem cell precursors but develop independently of the thymus. They comprise a key lymphocyte subset (approximately 10%-15% of peripheral blood mononuclear cells) and are a constituent of the innate immune system since these cells do not rearrange their germline DNA to obtain specificity. NK cells serve an important role in host defense against viral infections, as well as tumor surveillance. They are also a component of the adaptive immune response through cytokine production. NK cell functions are governed by a balance between activating receptors and inhibitory receptors.

NK cells are identified by expression of different cell-surface receptors and they are not a homogeneous population.(1) In general, the most common combination of surface markers used to identify the majority of NK cells is the absence of CD3 (CD3-), along with expression of CD56 (neural cell adhesion molecule) and CD16 (low-affinity IgG Fc receptor-Fc gamma RIII). However, not all NK cells express the CD56 and CD16 markers uniformly and, therefore, can be divided into subsets based on expression of these 2 molecules.(2) The CD16+ CD56+/- (dim or negative) that are CD3- are referred to as cytotoxic NK cells, while the CD56+ (bright) CD16- NK cells are called regulatory or cytokine secreting NK cells.(3) These are not only phenotypically and functionally distinct subsets but also developmentally separate. The majority of human NK cells (approximately 90%) have dim expression of CD56 and moderate to high levels of CD16, as well as perforin and granzymes (2 proteins mediating cytolytic activity) and are therefore high in cytotoxic capability. The remaining minority (approximately 10%) of NK cells are the CD56(bright) cytokine-producing NK cells. Therefore, cytotoxicity and cytokine production are the major functions of NK cells. Cytotoxicity can be subdivided into natural cytotoxicity directed largely toward virally infected cells or tumor cells, in the absence of prior stimulation or immunization, and antibody-dependent cellular cytotoxicity (ADCC) directed against antibody-coated target cells.(4) Circulating NK cells are enriched for the CD56(dim) phenotype, while within the lymph nodes, NK cells are largely CD56(bright). This differential localization is related to the pattern of homing receptors expressed on NK cells: CD56(dim) NK cells express homing markers for inflamed peripheral sites, while CD56(bright) NK cells express receptors for secondary lymphoid organs. The majority of circulating human NK cells, which have cytotoxic function and phenotype (CD56[dim]), are CD27-, while the CD56(bright) cells are CD27+. Therefore, the absence of CD27 expression identifies cytotoxic effector cells within the mature NK cell subsets.(5)

Natural killer T (NKT) cells represent a specialized T-cell population that is distinct from conventional T cells. They express an invariant T-cell receptor (TCR) that recognizes self and bacterial glycosphingolipid antigens presented by the MHC class I-like molecule, CD1d.(11) The development of NKT cells is also unique from regular T cells, as NKT cell precursors are positively selected by CD4+CD8+ cortical thymocytes and the signaling pathways differ from the conventional T cells. Activated NKT cells rapidly produce large amounts of Th1 and Th2 cytokines that transactivate other immune components and, therefore, NKT cells are involved in both innate and adaptive immune responses.(11)

NK cell deficiencies can be present as part of a larger immunological syndrome or as an isolated deficiency. Some of the primary (monogenic) immunodeficiencies that affect NK cell function or numbers include autoimmune lymphoproliferative syndrome (ALPS) related to CASP8 (caspase 8 variants); familial hemophagocytic lymphohistiocytosis (FHL) types 2, 3, and 4 due to variants in the PFP1 (encoding perforin), UNC13D (encoding the Munc13-4 protein) and STX-11 (encoding syntaxin -11), respectively; Hermansky-Pudlak syndrome (AP3B1); Papillon-Lefevre syndrome (CTSC: cathepsin C); nuclear factor kappa-beta essential modulator deficiency (NEMO) due to variants in the IKBKG gene; severe combined immunodeficiencies due to mutations in the IL-2RG, JAK3, ADA, PNP, ADK2 genes; bare lymphocyte syndrome (TAP2 gene); X-linked inhibitor of apoptosis protein deficiency (XIAP gene); X-linked lymphoproliferative disease (XLP): XLP-1 (due to variants in the SAP gene); Griscelli syndrome (RAB27A gene); Chediak-Higashi syndrome (LYST gene); and Wiskott-Aldrich syndrome (WAS gene).(12) Patients with X-linked inhibitor of apoptosis protein (XIAP) deficiency have been variably reported as having either normal numbers of NKT cells (13) or low numbers of NKT cells.(14) The apparent discrepancy in the numbers of NKT cells is likely related to the difference in size of the sample control groups and disease stage of patients between the 2 reports. At the present time, the role of XIAP in development of NKT cells has not been clearly delineated.

The isolated NK-cell deficiencies include the absolute NK-cell deficiency (ANKD), the classic NK-cell deficiency (CNKD), and the functional NK-cell deficiency (FNKD). NK-cell function is absent in ANKD and CNKD and deficient in FNKD, while NK cells are present in the latter but absent in the former 2 conditions. NKT cells are absent only in ANKD and present in both CNKD and FNKD.(12) NK-cell dysfunction has also been reported in systemic juvenile rheumatoid arthritis and macrophage activation syndrome.(15) There is also more data emerging on the pathogenic role of NK cells in atopic and autoimmune diseases.(4)

Patients with HIV-1 show a gradual loss of NK cells that correlates with disease progression. There is a selective loss of CD56(dim) NK cells, while the numbers of CD56(bright) NK cells remain the same. There appears to be a defect in differentiation from immature CD56- NK cells to mature CD56(dim) NK cells (16), with an expansion of the former (CD56-CD16+) NK cells in HIV viremic patients.(17) Differential mobilization of NK-cell subsets has also been reported related to acute exercise, with CD56(bright) NK cells being less responsive than CD56(dim) NK cells and the ratio of CD56(bright):CD56(dim) favors the former at least up to 1-hour post-exercise.(18)

NK cells also play an important role in regulating viral infections, and their deficiency predisposes individuals to susceptibility with herpes virus infections. NKG2D expression has been reported to decrease during human cytomegalovirus infection.(19) NK cells that express inhibitory receptors to self-MHC class I molecules are called "licensed," which means they are functionally more responsive to stimulation, while "unlicensed" NK cells lack receptors for self-MHC class I and are hyporesponsive. Contrary to the hypothesis that "licensed" NK cells are key for viral immunity, the depletion of "unlicensed" NK cells impairs control of viremia, suggesting that these cells are critical for protection against viral infection.

NK-cell lymphocytosis is seen in NK-neoplasias, extranodal NK/T-cell lymphoma, aggressive NK-cell leukemia, and blastic NK-cell lymphoma. Chronic NK-cell lymphocytosis is an indolent disorder characterized by proliferation of CD3-CD56+CD16- NK cells. Epstein-Barr virus (EBV) can infect nonneoplastic NK cells(20), and there is an expansion of CD16+CD56(dim) NK cells. Chronic active EBV infection involving NK cells can present with severe inflammatory and necrotic skin reactions typically associated with EBV+ NK-cell lymphoproliferative disease.(21)

The appropriate age-related reference values will be provided on the report. Pediatric reference values are not available for patients less than 7 years of age and therefore, interpretation will be based on the 7 to 17-year-old ranges with appropriate cautionary statements in the interpretation.

Interpretive comments will be provided, where applicable, along with reference range values for adult samples and pediatric samples 7 to 17 years of age. Since a separate pediatric reference range could not be established for patients less than 7 years of age at this time, interpretation of these samples will be made using the 7 to 17-year-old reference range as an approximate guideline.

Results should be interpreted in conjunction with the relevant clinical context and other appropriate immunological analyses.

1. Fan YY, Yang BY, Wu CY: Phenotypically and functionally distinct subsets of natural killer cells in human PBMCs. Cell Biol Int. 2008 Feb;32(2):188-197

2. Cooper MA, Fehniger TA, Caligiuri MA: The biology of human natural killer-cell subsets. Trends Immunol. 2001 Nov;22(11):633-640

3. Poli A, Michel T, Theresine M, Andres E, Hentges F, Zimmer J: CD56bright natural killer (NK) cells: an important NK cell subset. Immunology. 2009 Apr;126(4):458-465. doi: 10.1111/j.1365-2567.2008.03027.x

4. von Bubnoff D, Andres E, Hentges F, Bieber T, Michel T, Zimmer Jl: Natural killer cells in atopic and autoimmune diseases of the skin. J Allergy Clin Immunol. 2010 Jan;125(1):60-68. doi: 10.1016/j.jaci.2009.11.020

5. Vossen MT, Matmati M, Hertoghs KM, et al: CD27 defines phenotypically and functionally different human NK cell subsets. J Immunol. 2008 Mar;180(6):3739-3745. doi: 10.4049/jimmunol.180.6.3739

6. Suarez-Alvarez B, Lopez-Vazquez A, Baltar JM, Ortega F, Lopez-Larrea C: Potential role of NKG2D and its ligands in organ transplantation: a new target for immunointervention. Am J Transplant. 2009 Feb;9(2):251-257. doi: 10.1111/j.1600-6143.2008.02526.x

7. Borrego F, Robertson MJ, Ritz J, Pena J, Solana R: CD69 is a stimulatory receptor for NK cell and its cytotoxic effect is blocked by CD94 inhibitory receptor. Immunology. 1999 May;97(1):159-165. doi: 10.1046/j.1365-2567.1999.00738.x

8. Takahashi K, Aranami T, Endoh M, Miyake S, Yamamura T: The regulatory role of natural killer cells in multiple sclerosis. Brain. 2004 Sep;127(Pt 9):1917-1927. doi: 10.1093/brain/awh219

9. Alter G, Malenfant JM, Altfeld M: CD107a as a functional marker for the identification of natural killer cell activity. J Immunol Methods. 2004 Nov;294(1-2):15-22

10. Mathew PA, Chuang SS, Vaidya SV, Kumaresan PR, Boles KS, Pham HTK: The LLT1 receptor induces IFN-gamma production by human natural killer cells. Mol Immunol. 2004 Mar;40(16):1157-1163. doi: 10.1016/j.molimm.2003.11.024

11. Godfrey DI, Stankovic S, Baxter AG: Raising the NKT cell family. Nat Immunol. 2010 Mar;11(3):197-206. doi: 10.1038/ni.1841

12. Orange JS: Human natural killer cell deficiencies. Curr Opin Allergy Clin Immunol. 2006 Dec;6(6):399-409

13. Marsh RA, Villaneuva J, Kim MO, et al: Patients with X-linked lymphoproliferative disease due to BIRC4 mutation have normal invariant natural killer T-cell populations. Clin Immunol. 2009 Jul;132(1):116-123. doi: 10.1016/j.clim.2009.03.517

14. Rigaud S, Fondaneche MC, Lambert N, et al: XIAP deficiency in humans causes an X-linked lymphoproliferative syndrome. Nature. 2006 Nov 2;444:110-114. doi: 10.1038/nature05257

15. Villanueva J, Lee S, Giannini EH, et al: Natural killer cell dysfunction is a distinguishing feature of systemic onset juvenile rheumatoid arthritis and macrophage activation syndrome. Arthritis Res Ther. 2005;7:R30-R37. doi: 10.1186/ar1453

16. Tarazona R, Casado JG, Delarosa O, et al: Selective depletion of CD56(dim) NK cell subsets and maintenance of CD56(bright) NK cells in treatment-naive HIV-1-seropositive individuals. J Clin Immunol. 2002 May;22(3):176-183. doi: 10.1023/a:1015476114409

17. Mavilio D, Lombardo G, Benjamin J, et al: Characterization of CD56-/CD16+ natural killer (NK) cells: a highly dysfunctional NK subset expanded in HIV-infected viremic individuals. Proc Natl Acad Sci USA. 2005 Feb 22;102(8):2886-2891. doi: 10.1073/pnas.0409872102

18. Timmons BW, Cieslak T: Human natural killer subsets and acute exercise: a brief review. Exerc Immunol Rev. 2008;14:8-23

19. Muntasell A, Magri G, Pende D, Angulo A, Lopez-Botet M: Inhibition of NKG2D in NK cells by cytokines secreted in response to human cytomegalovirus infection. Blood. 2010 Jun 24;115(25):5170-5179. doi: 10.1182/blood-2009-11-256479

20. Trempat P, Tabiasco J, Andre P, et al: Evidence for early infection on nonneoplastic natural killer cells by Epstein-Barr virus. J Virol. 2002 Nov;76(21):11139-11142. doi: 10.1128/jvi.76.21.11139-11142.2002

21. Pacheco SE, Gottschalk SM, Gresik MV, Dishop MK, Okmaura T, McCormick TG: Chronic active Epstein-Barr virus infection of natural killer cells presenting as severe skin reaction to mosquito bites. J Allergy Clin Immunol. 2005 Aug;116(2):470-472. doi: 10.1016/j.jaci.2005.04.044

22. Mace EM, Orange JS: Emerging insights into human health and NK cell biology from the study of NK cell deficiencies. Immunol Rev. 2019;287(1):202-225

23. Delmonte OM, Fleisher TA: Flow cytometry: Surface markers and beyond. J Allergy Clin Immunol. 2019 Feb;143(2):528-537

24. Knight V, Heimall JR, Chong H, et al: A toolkit and framework for optimal laboratory evaluation of individuals with suspected primary immunodeficiency. J Allergy Clin Immunol Pract. 2021 Sep;9(9):3293-3307.e6

The absolute quantitation of natural killer (NK) subsets is performed as a 2-tube assay with BD Trucount tubes. Tube 1 provides the isotype control for CD56 and CD16 markers. A combination of antibodies directed against the following surface markers are used to identify the 4 major NK subsets as well as natural killer T  cells. Whole blood is stained with the surface marker antibodies in the dark for 20 minutes, followed by red blood cells lysis for 10 minutes in BD FACS Lysing solution. Samples are analyzed on a BD FACS Canto II flow cytometer and data analysis is performed in FACS DIVA 8.0 software.(Unpublished Mayo method)

Monday through Friday

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This test was developed using an analyte specific reagent. Its performance characteristics were determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the US Food and Drug Administration.

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