Test ID: NKCP
Natural Killer (NK) Cytotoxicity Profile

Natural Killer (NK) Cytotoxicity Profile

Assessment of patients with recurrent, severe herpes viral infections, primary or secondary hemophagocytic lymphohistiocytosis, and suspected or known monogenic defects affecting the NK cell compartment (approximately 30 known defects cause either functional or classic NK cell deficiency).

T- and B-Cell Quantitation by Flow Cytometry:

Monitoring CD4 counts and assessing immune deficiencies

When multiple specimen types are required to perform a panel of tests, the laboratory will perform the tests for which the appropriate specimen type was received and cancel those for which the appropriate specimen was not received. Please be advised that this may change the degree of interpretation received with the report.

NKC/81733: (51) Cr Release Assay

TBBS/9336: Flow Cytometry

Specimens must arrive within 24 hours of draw. Specimens are required to be received in the laboratory on weekdays by 2 p.m. No weekend processing. Ship specimen overnight in an Ambient Mailer-Critical Specimens Only (Supply T668).

Date and time of draw and ordering physician's name and phone number are required.

Note: The MML Expedite procedure is recommended to ensure specimens are processed as quickly as possible once received at Mayo Medical Laboratories. Contact 800-533-1710 to add this procedure to your order. A nominal fee will be charged.

Three separate specimens (a whole blood EDTA specimen and a whole blood heparin specimen in addition to a control specimen) are required.

Patient

Specimen Type: Whole blood

Container/Tube: Green top (sodium heparin)

Specimen Volume: 10 mL

Collection Instructions:

1. Send specimen in original tube. Do not aliquot.

2. Label specimen as "patient specimen."

Specimen Type: Whole blood

Container/Tube: Lavender top (EDTA)

Specimen Volume: 3 mL

Collection Instructions:

1. Send specimen in original tube. Do not aliquot.

2. Label specimen as "patient specimen."

Normal Control

Specimen Type: Whole blood

Container/Tube: Green top (sodium heparin)

Specimen Volume: 10 mL

Collection Instructions:

1. Draw a control blood from a normal (healthy),unrelated person at the same time as the patient.

2. Send specimen in original tube. Do not aliquot.

3. Label clearly on outermost label "normal or shipping control."

4. Rubberband with patient specimen.

Natural Killer (NK) Cytotoxicity Profile

Natural killer (NK) cell cytotoxicity includes spontaneous cytotoxicity against target cells, typically lacking surface major histocompatibility complex (MHC) class I expression, cytokine-stimulated cytotoxicity (also called lymphokine-associated cytotoxicity [LAK]), and antibody-dependent cellular cytotoxicity (ADCC). NK cell cytotoxic function is a key aspect of innate immunity and critical to maintenance of immune function, particularly with regard to viral infections (herpes viruses) and tumor surveillance. Cytotoxic NK cells typically express CD16 at high levels and CD56 at very low levels (CD16+56+) and account for the majority of circulating NK cells, while cytokine-producing NK cells express CD56 abundantly but little to no CD16 (CD56brightCD16+/-) expression. Cytotoxic NK cells contain cytotoxic granules expressing proteins such as perforin, Granzyme A and B and Granulysin, which participate in the effector cytotoxic function.   

T- and B-Cell Quantitation by Flow Cytometry

Normal immunity requires a balance between the activities of various lymphocyte subpopulations with different effector and regulatory functions.

Different immune cells can be characterized by unique surface membrane antigens described by a cluster of differentiation nomenclature (eg, CD3 is an antigen found on the surface of T lymphocytes). Abnormalities in the number and percent of T (CD3), T-helper (CD4), T-suppressor (CD8), B (CD19), and natural killer (CD16+CD56) lymphocytes have been described in a number of different diseases. In patients who are infected with HIV, the CD4 count is measured for AIDS diagnosis and for initiation of antiviral therapy. The progressive loss of CD4 T lymphocytes in patients infected with HIV is associated with increased infections and complications.

The Public Health Service has recommended that all HIV-positive patients be tested every 3 to 6 months for the level of CD4 T lymphocytes.

The absolute counts of lymphocyte subsets are known to be influenced by a variety of biological factors, including hormones, the environment, and temperature. The studies on diurnal (circadian) variation in lymphocyte counts have demonstrated progressive increase in CD4 T-cell count throughout the day, while CD8 T cells and CD19+ B cells increase between 8:30 am and noon, with no change between noon and afternoon. NK cell counts, on the other hand, are constant throughout the day.(1) Circadian variations in circulating T-cell counts have been shown to be negatively correlated with plasma cortisol concentration.(2-4) In fact, cortisol and catecholamine concentrations control distribution and, therefore, numbers of naive versus effector CD4 and CD8 T cells.(2) It is generally accepted that lower CD4 T-cell counts are seen in the morning compared with the evening (5), and during summer compared to winter.(6)

These data, therefore, indicate that timing and consistency in timing of blood collection is critical when serially monitoring patients for lymphocyte subsets.

The appropriate age-related reference values will be provided on the report.

Cytotoxic activity (% killing) is reported for a series of titrating effector (E) to target (T) ratios. Reference values for each E:T ratio obtained as an average of a cohort of healthy individuals is provided with the report. 

T- and B-Cell Quantitation by Flow Cytometry

When the CD4 count falls below 500 cells/mcL, HIV-positive patients can be diagnosed with AIDS and can receive antiretroviral therapy.

When the CD4 count falls below 200 cells/mcL, prophylaxis against Pneumocystis jiroveci pneumonia is recommended.

A single abnormal or low result for natural killer (NK) cytotoxic function should not be interpreted as being indicative of NK cell functional deficiency.

At least 3 consecutive results should be obtained to verify functional NK cell deficiency.  

It is useful to simultaneously perform total NK cell quantitation in blood to ensure that there is no quantitative NK cell deficiency that can result in low function.  

There can be considerable variability in NK cell function between individuals and within an individual over time.  

Steroids as well as other immunosuppressants may affect NK cell function.

This test should not be ordered as part of a routine immunological evaluation as a first-tier test. Only patients with specific clinical indications where assessment of NK cell function is warranted should be tested with this assay (described in the clinical information and useful for sections).

NK cell cytotoxic activity diminishes with time in heparinized blood specimens regardless of storage conditions. Specimens >24 hours old may yield falsely low results. Therefore, samples should be ideally tested within 24 hours of blood collection.

Approximately 25% of healthy individuals may present at any given time with decreased NK cell cytotoxic function, which is why a minimum of 3 consecutive results is required to establish true NK cell functional deficiency and correlation must be made with clinical phenotype.

T- and B-Cell QN by Flow Cytometry This assay is not used for diagnosing lymphocytic malignancies or evaluation of lymphocytosis of unknown etiology. In these situations, LCMS/3287 Leukemia/Lymphoma Immunophenotyping by Flow Cytometry, which includes a hematopathology review, should be ordered.

Timing and consistency in timing of blood collection is critical when serially monitoring patients for lymphocyte subsets. See Clinical Information.

Natural Killer (NK) Cytotoxicity Profile

Patarca R, Fletcher MA, Podack ER: Chapter 33: Cytolytic cell functions. In Manual of Clinical Laboratory Immunology. Fifth edition. Edited by NR Rose, E Conway de Macario, JD Folds, et al. Washington, DC, ASM Press, 1997

T- and B-Cell Quantitation by Flow Cytometry:

1. Carmichael KF, Abayomi A: Analysis of diurnal variation of lymphocyte subsets in healthy subjects and its implication in HIV monitoring and treatment. 15th Intl Conference on AIDS, Bangkok, Thailand, 2004, Abstract B11052

2. Dimitrov S, Benedict C, Heutling D, et al: Cortisol and epinephrine control opposing circadian rhythms in T-cell subsets. Blood 2009 (prepublished online March 17, 2009)

3. Dimitrov S, Lange T, Nohroudi K, Born J: Number and function of circulating antigen presenting cells regulated by sleep. Sleep 2007;30:401-411

4. Kronfol Z, Nair M, Zhang Q, et al: Circadian immune measures in healthy volunteers: relationship to hypothalamic-pituitary-adrenal axis hormones and sympathetic neurotransmitters. Psychosom Med 1997;59:42-50

5. Malone JL, Simms TE, Gray GC, et al: Sources of variability in repeated T-helper lymphocyte counts from HIV 1-infected patients: total lymphocyte count fluctuations and diurnal cycle are important. J AIDS 1990;3:144-151

6. Paglieroni TG, Holland PV: Circannual variation in lymphocyte subsets, revisited. Transfusion 1994;34:512-516

7. Mandy FF, Nicholson JK, McDougal JS: Guidelines for performing single-platform absolute CD4+T-cell determinations with CD45 gating for persons infected with human immunodeficiency virus. Center for Disease Control and Prevention. MMWR Morb Mortal Wkly Rep 2003;52:1-13

8. Centers for Disease Control: 1997 Revised guidelines for performing CD4+ T-cell determinations in persons infected with human immunodeficiency virus (HIV). MMWR Morb Mortal Wkly Rep 46 no. RR-2: 1997, pp 1-29

9. U.S. Department of Health and Human Services: Recommendations for prophylaxis against Pneumocystis carinii pneumonia for adults and adolescents infected with human immunodeficiency virus. MMWR Morb Mortal Wkly Rep 43 no. RR-3: 1994, pp 1-21

Natural Killer (NK) Cytotoxicity Profile

NK cell cytotoxic functional activity is estimated by a standard 4-hour stimulation of effector cells (patient or control peripheral blood mononuclear cells) incubated with target cells (monocytic cell K562 lacking major histocompatibility complex class I expression) labeled with 51Cr. The % cytotoxicity (51Cr release) is measured for a series of effector to target concentrations. Maximal cytotoxicity is measured using a detergent for target cell lysis as an experimental control and spontaneous cytotoxicity in the absence of effector stimulation is also measured.(Unpublished Mayo method)  

T- and B-Cell Quantitation by Flow Cytometry

The T- and B-cell surface marker assay uses monoclonal antibodies to identify the various membrane antigens, and flow cytometry to enumerate the number of cells expressing these differentiation antigens. The results are reported as the percent of lymphocytes that are T-helper (CD3+, CD4+), T-suppressor (CD3+, CD8+), natural killer (CD16+56+, CD3-), and B-lymphocytes (CD19+), and the absolute number of each cell type per mL of blood. The assay is a 4-color, no-wash procedure and the absolute counts are calculated from internal bead standards. In addition, the total lymphocyte count and the helper-suppressor ratio (T[H]/T[S]) is reported.(Hoffman RA, Kung PC, Hansen WP, Goedstien G: Simple and rapid measurement of human T lymphocytes and their subclasses in peripheral blood. Proc Natl Acad Sci USA, 1980:77:4914-4917; US Department of Health and Human Services: Guidelines for performance of CD4+ T-cell determinations in persons with human immunodeficiency virus infection. MMWR Morb Mortal Wkly Rep 1997;46[no. RR-2]:1-29)

N.K. Cytotoxicity: Monday through Friday; Continuously to 2 p.m.

T- and B-Cell QN by Flow Cytometry: Monday through Sunday

86355-B cells, total count

86357-Natural killer (NK) cells, total count

86359-T cells, total count

86360-Absolute CD4/CD8 count with ratio

86805-Lymphocytotoxicity assay, visual crossmatch; with titration