Test ID TCRVB TCR V-Beta Repertoire Analysis by Spectratyping, Blood
Useful For
Assessment of T-cell receptor diversity in various clinical contexts including primary immunodeficiencies, monitoring immune reconstitution posthematopoietic cell transplantation, and temporal assessment of repertoire changes in autoimmune diseases and viral infections
Testing Algorithm
Additional tests that could be ordered include:
-TREC / T-Cell Receptor Excision Circles (TREC) Analysis, Blood
-CD4RT / CD4 T-Cell Recent Thymic Emigrants (RTE)
-TCP / T-Cell Subsets, Naive, Memory, and Activated
Special Instructions
Method Name
Molecular TCR Vb-CDR3 Fragment Length Analysis
Reporting Name
TCR Spectratyping, BSpecimen Type
Whole Blood EDTAAdvisory Information
Mayo Laboratory Director/Consultant approval is required prior to ordering this test in patients >40 years of age.
Shipping Instructions
It is recommended that specimens arrive within 24 hours of draw.
Samples arriving on the weekend and observed holidays may be canceled.
Necessary Information
Ordering physician's name and phone number are required.
Specimen Required
For serial monitoring, it is recommended to perform specimen collection at the same time of day, if possible.
Supplies: Ambient Shipping Box-Critical Specimens Only (T668)
Specimen Type: Blood
Container/Tube: Lavender top (EDTA)
Specimen Volume:
Adults: 10 mL
Pediatrics:
-Preferred volume for >1 year: 3 mL
-Preferred volume for ≤1 year: 1 mL
Collection Instructions: Send specimen in original tube.
Specimen Minimum Volume
Adults: 5 mL
Pediatrics: 1 mL
Specimen Stability Information
Specimen Type | Temperature | Time | Special Container |
---|---|---|---|
Whole Blood EDTA | Ambient | 48 hours | PURPLE OR PINK TOP/EDTA |
Clinical Information
The rearrangement of the T-cell receptor (TCR) through somatic recombination of V (variable), D (diversity), J (joining), and C (constant) regions is a defining event in the development and maturation of a T cell. TCR gene rearrangement takes place in the thymus. During the process of rearrangement, DNA byproducts are generated called T-cell receptor excision circles (TRECs) and these are used as markers of T cells that have recently emigrated from the thymus (TREC / T-Cell Receptor Excision Circles [TREC] Analysis, Blood). T cells, as part of the adaptive immune system, recognize foreign antigens when they are displayed on the surface of the body's own cells. T cells recognize these foreign antigens as peptides presented in the context of major histocompatibility complex (MHC) molecules through their T-cell receptors. Each TCR exists as 2 different polypeptide chains (heterodimers) called the TCR alpha chain and TCR beta chain, and these are linked by disulfide bonds. The majority of T cells (approximately 90%) in the body express TCRs with alpha and beta chains. A minority of T cells express other T-cell receptors made of different polypeptide chains, gamma and delta. Each T cell has approximately 30,000 identical antigen receptors on its cell surface. A TCR has only 1 antigen-binding site, in contrast to the B-cell receptor, which has 2, and TCRs are never secreted and always remain on the cell surface. The alpha and beta chains are encoded by different gene loci (alpha and beta TCR gene locus). The beta chain locus rearranges before the alpha chain and a functional beta chain has to be produced in order for the T cell to form a pre-T-cell receptor. The expression of the rearranged beta chain with an alpha chain precursor suppresses additional gene rearrangement at the TCR beta locus. The TCR alpha chain locus rearrangement can proceed even with production of a functional alpha chain until there is positive selection of the particular T cell. However, it is important to note that each T cell has a single functional specificity for its TCR.
A key concept in understanding the immune response is that there is enormous diversity in the immune system to enable protection against a huge array of pathogens. Since the germline genome is limited in size, diversity is achieved not only by the process of V(D)J recombination but also by junctional (junctions between V-D and D-J segments) deletion of nucleotides and addition of pseudo-random, nontemplated nucleotides. In particular, the CDR3 (complementarity determining region 3), which is the most critical determinant of antigenic specificity in T cells (and also B cells) is short (between 66-90 nucleotides, approximately 20-30 amino acids) and amenable to assessment of length by fragment length analysis, which provides a size resolution of up to 1 base pair between different CDR3 regions. It is thought that the CDR3-TCR beta chain repertoire in healthy adults contains somewhere between 3 and 4 million unique sequences.(1) Other reports suggest that the unique TCR repertoire after thymic selection is between 10 to 100 million in humans.(2) There is, however, a bias in TCR selection with overrepresentation of certain TCRs that are widely used in individuals who share the same major histocompatibility (MHC) types and these are called "public TCRs." Public TCRs generally have fewer random nucleotide additions in their sequence. The TCR V beta repertoire varies significantly between individuals and populations because of 7 frequently occurring inactivating polymorphisms in functional gene segments and a large insertion/deletion-related polymorphism encompassing 2 V beta gene segments. With this latter situation, the TCR Vb 6-2/6-3 and TCR Vb 4-3 genes are frequently deleted from all ethnic groups.(3) It has been reported that the total number of functional TCR V beta gene segments expressed by an individual varies from 42 to 47.(4)
Deep sequencing technologies are evolving to analyze this large diversity in the adaptive immune receptors,(5,6) however; deep sequencing of the T-cell and B-cell receptor genes is not yet widely available and is expensive. Flow cytometry-based analysis to assess TCR V beta diversity is available; however, the antibodies are limited and therefore the assay is not capable of assessing the entire TCR V beta repertoire. On the other hand, TCR beta chain repertoire analysis by fragment length analysis (spectratyping) using fluorescent primers to measure CDR3 length variability, while unable to provide the extreme high resolution of deep sequencing, can provide a global "snapshot" of T-cell receptor repertoire diversity, which is useful for most clinical applications where this level of assessment is required.(7-14) It is important to note that this method uses PCR to amplify the rearranged variable regions to provide adequate template for sequencing (fragment length analysis), and this can introduce bias due to the more efficient amplification of certain templates compared to others. However, despite this limitation, since this assay is not quantitative, it is still able to provide an assessment of diversity by measuring the CDR3 length in various TCR V beta genes, which are organized into 24 families.
Reference Values
References values will be provided in the patient report.
Interpretation
An interpretive report will be provided with adult and pediatric reference values for the relative contribution of each family to the total repertoire (% diversity ratio). The interpretation will be based on visual analysis of the spectratype (polyclonal, oligoclonal, or monoclonal) for each family as well as assessment of the number of peaks (numerical value not reported), and diversity ratio (DR) (reported value). Information on the distribution of peaks, eg, Gaussian vs non-Gaussian, will also be included in the report, where appropriate. Internal analytical and quality controls will be assessed to determine the suitability of reporting a patient result. Correlation with the clinical context will be made when possible, based on clinical history provided in the patient information sheet (which should be provided with the patient sample).
Clinical Reference
1. Robins HS, Campregher PV, Srivastava SK, et al: Comprehensive assessment of T-cell receptor beta-chain diversity in alphabeta T cells. Blood 2009;114:4099-4107
2. Arstila TP, Casrouge A, Baron V, et al: A direct estimate of the human alpha-beta T cell receptor diversity. Science 1999;958-961
3. Brennan RM, Petersen J, Neller MA: The impact of a large and frequent deletion in the human TCR beta locus on antiviral immunity. J Immunol 2012;188:2742-2748
4. Mackelprang R, Carlson CS, Subrahmanyan L, et al: Sequence variation in the human T-cell receptor loci. Immunol Rev 2002;190:26-39
5. Warren EH, Matsen IV FA, Chou J: High-throughput sequencing of B- and T-lymphocyte antigen receptors in hematology. Blood 2013;122:19-22
6. Robins H: Immunosequencing: applications of immune repertoire deep sequencing. Curr Opin Immunol 2013;25:646-652
7. Gorski J, Yassai M, Zhu X, et al: Circulating T cell repertoire complexity in normal individuals and bone marrow recipients analyzed by CDR3 spectratyping. J Immunol 1994;152:5109-5119
8. Memon SA, Sportes C, Flomerfelt FA, et al: Quantitative analysis of T cell receptor diversity in clinical samples of human peripheral blood. J Immunol Methods 2012;375:84-92
9. van Heijst JMJ, Ceberio I, Lipuma LB, et al: Quantitative assessment of T cell repertoire recovery after hematopoietic stem cell transplantation. Nature Medicine 2013;19:372-378
10. Wada T, Schurman SH, Garabedian EK, et al: Analysis of T-cell repertoire diversity in Wiskott-Aldrich syndrome. Blood 2005;106:3895-3897
11. Pirovano S, Mazzolari E, Pasic S, et al: Impaired thymic output and restricted T-cell repertoire in two infants with immunodeficiency and early-onset generalized dermatitis. Immunol Letters 2003;86:93-97
12. Villa A, Notarangelo LD, Roifman CM: Omenn syndrome: inflammation in leaky severe combined immunodeficiency. J Allergy Clin Immunol 2008;122:1082-1086
13. Sullivan KE: The clinical, immunological and molecular spectrum of chromosome 22q11.2 deletion syndrome and DiGeorge syndrome. Curr Opin Allergy Clin Immunol 2004;4:505-512
14. Markert ML, Devlin BH, McCarthy EA: Thymus transplantation. Clin Immunol 2010;135:236-246
Day(s) and Time(s) Performed
Varies
Do not draw specimen after Thursday. Specimen must be received by 10 a.m. on Friday.
Analytic Time
20 daysTest Classification
This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the U.S. Food and Drug Administration.CPT Code Information
81340-TRG (T cell antigen receptor, beta) (eg, leukemia and lymphoma), gene rearrangement analysis to detect abnormal clonal population(s); using amplification methodology (eg, polymerase chain reaction)
LOINC Code Information
Test ID | Test Order Name | Order LOINC Value |
---|---|---|
TCRVB | TCR Spectratyping, B | In Process |
Result ID | Test Result Name | Result LOINC Value |
---|---|---|
36473 | Family 2 Diversity Ratio | 82501-8 |
36474 | Family 3-1 Diversity Ratio | 82500-0 |
36475 | Family 4 Diversity Ratio | 82499-5 |
36476 | Family 5 Diversity Ratio | 82498-7 |
36477 | Family 6 Diversity Ratio | 82497-9 |
36478 | Family 7 Diversity Ratio | 82496-1 |
36479 | Family 9 Diversity Ratio | 82495-3 |
36480 | Family 10 Diversity Ratio | 82494-6 |
36481 | Family 11 Diversity Ratio | 82493-8 |
36482 | Family 12 Diversity Ratio | 82492-0 |
36483 | Family 13 Diversity Ratio | 82491-2 |
36484 | Family 14 Diversity Ratio | 82490-4 |
36485 | Family 15 Diversity Ratio | 82489-6 |
36486 | Family 16 Diversity Ratio | 82488-8 |
36487 | Family 18 Diversity Ratio | 82487-0 |
36488 | Family 19 Diversity Ratio | 82486-2 |
36489 | Family 20-1 Diversity Ratio | 82485-4 |
36490 | Family 24-1 Diversity Ratio | 82484-7 |
36491 | Family 25 Diversity Ratio | 82483-9 |
36492 | Family 27 Diversity Ratio | 82482-1 |
36493 | Family 28 Diversity Ratio | 82481-3 |
36494 | Family 29 Diversity Ratio | 82480-5 |
36518 | Family 30 Diversity Ratio | 82479-7 |
36495 | Interpretation | 69047-9 |
36496 | Reviewed by | 18771-6 |
Forms
TCR V beta Spectratyping Assay Patient Information (T719) in Special Instructions