Test ID: TREGS
T-Cell Subsets, Regulatory (Tregs)
NY State Approved 
Indicates the status of NY State approval and if the test is orderable for NY State clients.
Useful For Suggests clinical disorders or settings where the test may be helpful
Evaluating patients with clinical features of IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked inheritance) and other primary immunodeficiencies, autoimmune diseases, allergy and asthma, and graft-vs-host disease post-hematopoietic stem cell transplantation
Method Name A short description of the method used to perform the test
Flow Cytometry
Reporting Name A shorter/abbreviated version of the Published Name for a test; an abbreviated test name
Aliases Lists additional common names for a test, as an aid in searching
Autoimmunity
FOXP3 mutation
FOXP3+ T-cells
GVHD
IPEX
Natural naive Tregs
Natural Tregs
Regulatory T-cells
STAT5b mutation
Tr1/Th3 T-cells
Tregs
Specimen Type Describes the specimen type needed for testing
Specimen Required Defines the optimal specimen. This field describes the type of specimen required to perform the test and the preferred volume to complete testing. The volume allows automated processing, fastest throughput and, when indicated, repeat or reflex testing.
Send specimen Monday through Thursday only. Specimen must arrive within 24 hours of draw and by 10 a.m. on Friday. Draw and package specimen as close to shipping time as possible. Ship specimen overnight.
Container/Tube: Lavender top (EDTA)
Specimen Volume: 3 mL
Collection Instructions: Send specimen in original tube. Do not aliquot.
Additional Information:
1. Ordering physician's name and phone number are required.
2. For serial monitoring, we recommend that specimen draws be performed at the same time of day.
Specimen Minimum Volume Defines the amount of specimen required to perform an assay once, including instrument and container dead space. Submitting the minimum specimen volume makes it impossible to repeat the test or perform confirmatory or perform reflex testing. In some situations, a minimum specimen volume may result in a QNS (quantity not sufficient) result, requiring a second specimen to be collected.
Reject Due To Identifies specimen types and conditions that may cause the specimen to be rejected
| Hemolysis | Mild OK; Gross reject |
| Lipemia | Mild OK; Gross reject |
| Icterus | NA |
| Other | NA |
Specimen Stability Information Provides a description of the temperatures required to transport a specimen to the laboratory. Alternate acceptable temperature(s) are also included.
| Specimen Type | Temperature | Time |
|---|---|---|
| Whole Blood EDTA | Ambient | 48 hours |
Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test
Regulatory T cells (Tregs) are a population of CD4+ T cells with a unique role in the immune response. Tregs are crucial in suppressing aberrant pathological immune responses in autoimmune diseases, transplantation, and graft-vs-host disease after allogeneic hematopoietic stem cell transplantation.(1) Tregs are activated through the specific T-cell receptor, but their effector function is nonspecific and they regulate the local inflammatory response through cell-to-cell contact and cytokine secretion.(2) Tregs secrete interleukin (IL)-9 (IL-9), IL-10, and transforming growth factor-beta 1 (TGF-beta 1), which aid in the mediation of immunosuppressive activity.
Chief characteristics of the Treg population are surface expression of the CD25 protein (IL-2Ra) and the intracellular presence of the transcription factor Foxp3. The IL-7 receptor (CD127) is downregulated on Foxp3+CD4+CD25+ T cells and provides an excellent alternative cell-surface marker to Foxp3 for detecting natural Tregs (CD4+CD25+CD127lo).(2)
Natural Tregs account for 5% to 10% of the total CD4 T-cell population and are derived from thymic precursors.(3) Since CD25 is also expressed on activated T cells, the concomitant use of CD127 permits the differentiation of Tregs from activated T cells.(4) Natural Tregs express the memory marker CD45RO and have limited ability to proliferate. However, within the CD4+CD25+Treg population, there is a subset of Tregs that express the CD45 isoform generally associated with naive T cells (CD45RA), and this subset has been called natural naive (Nn) Tregs. Nn Tregs are most prominent in young adults and decrease with age along with the rest of the naive CD4 T-cell population.(5) Like other naive T cells, Nn Tregs have high proliferative capacity, as well as the suppressor activity of other Treg subsets. Present evidence suggests that Nn Tregs also have a thymic ancestry and are the precursors of the natural Tregs (that are of the memory, antigen-experienced phenotype) and appear to be composed of T cells with self-reactive T-cell receptors.(5)
Other subsets of Tregs include the Th3 cells, which secrete high levels of TGF-beta 1 and can be induced by oral administration of antigen, and regulatory T class 1 (Tr1) cells, which secrete interferon-gamma and IL-10.(5) These Treg subsets are most likely induced in the periphery and are responsible for peripheral tolerance to self antigens. The suppressive activity of Th3 and Tr1 cells are related to the cytokines they produce, TGF-beta 1 and IL-10, respectively.
The absence of Tregs as a result of mutations in the FOXP3 gene cause a primary immunodeficiency called IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked inheritance).(6) Patients with IPEX have a complex manifestation of symptoms including severe watery diarrhea due to significant villous atrophy and lymphocytic infiltration of bowel mucosa, early-onset autoimmune endocrinopathies involving the pancreas or thyroid, and a dermatitic (eczematous) rash. In addition, there are other autoimmune manifestations including autoimmune cytopenias and autoimmune hepatitis. Renal disease is quite common in these patients. Finally, these patients also have a significant predisposition to infections including sepsis, pneumonia, meningitis, and osteomyelitis.(6) Decreased Foxp3+CD4+CD25+Tregs have been reported in 1 patient with a STAT5b mutation.(7)
There is an expansion of Nn Tregs in patients with monoclonal gammopathy of undetermined significance and multiple myeloma, likely as a response to the process of malignant transformation.(8) Expansion of Tregs has also been reported in other neoplasias including B-cell chronic lymphocytic leukemia, Hodgkin disease, and solid tumors.
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. Natural killer cell counts, on the other hand, are constant throughout the day.(9) Circadian variations in circulating T-cell counts have been shown to be negatively correlated with plasma cortisol concentration.(10-12) In fact, cortisol and catecholamine concentrations control distribution and, therefore, numbers of naive versus effector CD4 and CD8 T cells.(10) It is generally accepted that lower CD4 T-cell counts are seen in the morning compared with the evening,(13) and during summer compared to winter.(14) These data, therefore, indicate that timing and consistency in timing of blood collection is critical when serially monitoring patients for lymphocyte subsets.
Reference Values Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.
The appropriate age-related reference values will be provided on the report.
Interpretation Provides information to assist in interpretation of the test results
The lack of regulatory T (Treg) cells is associated with mutations in the FOXP3 gene. Low Tregs are also seen in the context of STAT5b mutations. Reduced Nn Tregs and natural Tregs are likely to predispose to autoimmunity, while reductions in Th3/Tr1 cells may impair oral and peripheral tolerance, also facilitating the development of autoimmunity.
The presence of expanded naive Tregs may indicate a process of malignant transformation, if other clinical features of malignant disease are present.
Increased Tregs in donor stem cell allografts have been associated with a reduced incidence of graft-versus-host disease (ie, mediating a protective effect) after allogeneic stem cell transplantation.
Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances
This panel provides only quantitative information regarding the various regulatory T cell (Treg) subsets; it does not provide information on the functional aspect of these populations.
Results should be correlated with clinical presentation.
Molecular testing is required to confirm a diagnosis of IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked inheritance). Contact Mayo Medical Laboratories at 800-533-1710 for assistance in ordering molecular testing.
Treg cells may be reduced in a variety of clinical contexts such as in autoimmune diseases or allograft rejection.
Timing and consistency in timing of blood collection is critical when serially monitoring patients for lymphocyte subsets. See data under Clinical Information.
Clinical Reference Provides recommendations for further in-depth reading of a clinical nature
1. Sakaguchi S, Sakaguchi N, Shimizu J, et al: Immunologic tolerance maintained by CD25+CD4+ regulatory T-cells: their common role in controlling autoimmunity, tumor immunity, and transplantation tolerance. Immunol Rev 2001;182:18-32
2. Liu W, Putnam A, Xu-yu Z, et al: CD127 expression inversely correlates with FOXP3 and suppressive function of human CD4+ Treg cells. J Exp Med 2006;203(7):1701-1711
3. Seddiki N, Santner-Nanan B, Tangye SG, et al: Persistence of naive CD45RA+ regulatory T-cells in adult life. Blood 2006;107:2830-2838
4. Seddiki N, Santner-Nanan B, Martinson J, et al: Expression of IL-2 and IL-7 receptors discriminates between human regulatory and activated T-cells. J Exp Med 2006;203(7):1693-1700
5. Valmori D, Merlo A, Souleimanian NE, et al: A peripheral circulating compartment of natural naive CD4+Tregs. J Clin Invest 2005;115(7):1953-1962
6. Torgerson TR, Ochs HD: IPEX: FOXP3 mutations and lack of regulatory T-cells. J Allergy Clin Immunol 2007;120:744-750
7. Cohen AC, Nadeau KC, Tu W, et al: Decreased accumulation and regulatory function of CD4+CD25 (high) T-cells in human STAT5b deficiency. J Immunol 2006;177:2770-2774
8. Beyer M, Kochanek M, Giese T, et al: In vivo peripheral expansion of naive CD4+CD25(high) FOXP3+ regulatory T-cells in patients with multiple myeloma. Blood 2006;107:3940-3949
9. 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
10. Dimitrov S, Benedict C, Heutling D, et al: Cortisol and epinephrine control opposing circadian rhythms in T-cell subsets. Blood 2009;113(21):5134-5143
11. Dimitrov S, Lange T, Nohroudi K, Born J: Number and function of circulating antigen presenting cells regulated by sleep. Sleep 2007;30:401-411
12. 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
13. 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
14. Paglieroni TG, Holland PV: Circannual variation in lymphocyte subsets, revisited. Transfusion 1994;34:512-516
Method Description Describes how the test is performed and provides a method-specific reference
EDTA anticoagulated blood is incubated with antibodies to various T-cell markers (ie, CD4, CD127, CD45RO, CD45RA, and CD25). After RBC lysis, the sample is washed to remove any unbound antibodies prior to analysis on a BD FACS CantoA (6-color) flow cytometry instrument. The assay uses 2 antibody tubes for data acquisition, but analysis is performed as a single panel. Each Treg subset is expressed as a percentage of total CD4+ T cells. BD Tru Count tubes are used to calculate the absolute count of cells per mcL for each subset. The regulatory T-cell panel is linked to the TCD4 test (TCD4/84348 CD4 Count for Immune Monitoring, Blood) within the experiment and, therefore, the CD3, CD4, and CD8 T-cell reference ranges are provided within the TCD4 assay. The regulatory T cell results are interpreted using a reference range derived from data of normal healthy adult and pediatric donors. Isotype controls are used in each assay to measure background fluorescence of the samples. A normal, healthy control is also included in each experiment to ensure the optimal performance of the assay.(Unpublished Mayo information)
Day(s) and Time(s) Test Performed Outlines the days and times the test is performed. This field reflects the day and time the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time required before the test is performed. Some tests are listed as continuously performed, which means assays are performed several times during the day.
Monday through Friday
Do not send specimen after Thursday. Specimen must be received by 10 a.m. on Friday.
Analytic Time Defines the amount of time it takes the laboratory to setup and perform the test. This is defined in number of days. The shortest interval of time expressed is "same day/1 day," which means the results may be available the same day that the sample is received in the testing laboratory. One day means results are available 1 day after the sample is received in the laboratory.
Maximum Laboratory Time Defines the maximum time from specimen receipt at Mayo Medical Laboratories until the release of the test result
Specimen Retention Time Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded
Performing Laboratory Location The location of the laboratory that performs the test
Test Classification Provides information regarding the medical device classification for laboratory test kits and reagents. Tests may be classified as cleared or approved by the US Food and Drug Administration (FDA) and used per manufacturer's instructions, or as products that do not undergo full FDA review and approval, and are then labeled as an Analyte Specific Reagent (ASR), Investigation Use Only (IUO) product, or a Research Use Only (RUO) product.
CPT Code Information Provides guidance in determining the appropriate Current Procedural Terminology (CPT) code(s) information for each test or profile. The listed CPT codes reflect Mayo Medical Laboratories interpretation of CPT coding requirements. It is the responsibility of each laboratory to determine correct CPT codes to use for billing.
86359-T cells, total count
86361-Absolute CD4 count
LOINC® Code Information Provides guidance in determining the Logical Observation Identifiers Names and Codes (LOINC) values for the result codes returned for this test or profile.
| Result ID | Reporting Name | LOINC Code |
|---|---|---|
| 29143 | % Activated CD4+ T cells (4+CD25+) | 13332-2 |
| 29144 | % Natural Tregs | In Process |
| 29145 | % N. Naive Tregs | In Process |
| 29146 | % CD4+CD25-CD127+ (Tr1/Th3) | In Process |
| 29147 | Activated CD4+ T cells (4+CD25+) | 26982-9 |
| 29148 | CD4+CD25+CD127loCD45RO+ (Nat Tregs) | In Process |
| 29149 | CD4+CD25+CD127loCD45RA+ Naive Tregs | In Process |
| 29150 | CD4+CD25-CD127+ (Tr1/Th3) | In Process |
| 29177 | Interpretation | 69052-9 |
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