Test Catalog

Test Id : TCRVB

TCR V-Beta Repertoire Analysis by Spectratyping, Blood

Useful For
Suggests clinical disorders or settings where the test may be helpful

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
Delineates situations when tests are added to the initial order. This includes reflex and additional tests.

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
Library of PDFs including pertinent information and forms related to the test

Method Name
A short description of the method used to perform the test

Molecular TCR Vb-CDR3 Fragment Length Analysis

NY State Available
Indicates the status of NY State approval and if the test is orderable for NY State clients.

Yes

Reporting Name
Lists a shorter or abbreviated version of the Published Name for a test

TCR Spectratyping, B

Aliases
Lists additional common names for a test, as an aid in searching

Bone marrow transplantation

DiGeorge Syndrome

Hematopoietic cell transplantation

Immune Reconstitution

Immune System Diversity

Immunological recovery

Leaky SCID

Oligoclonal T cells

Omenn Syndrome

Primary Immunodeficiency

SCID

Spectratyping

T cell diversity

T cell recovery

T Cell Repertoire

T cell spectratype

TCR V-beta

Testing Algorithm
Delineates situations when tests are added to the initial order. This includes reflex and additional tests.

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

Specimen Type
Describes the specimen type validated for testing

Whole Blood EDTA

Ordering Guidance

Mayo Laboratory Director/Consultant approval is required prior to ordering this test in patients >40 years of age.

Shipping Instructions

Specimens are required to be received in the laboratory weekdays and by 4 p.m. on Friday. Draw and package specimen as close to shipping time as possible.

 

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
Defines the optimal specimen required to perform the test and the preferred volume to complete testing

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 < or =1 year: 1 mL

Collection Instructions: Send specimen in original tube.

Special Instructions
Library of PDFs including pertinent information and forms related to the test

Forms

Specimen Minimum Volume
Defines the amount of sample necessary to provide a clinically relevant result as determined by the Testing Laboratory

Adults: 5 mL

Pediatrics: 1 mL

Reject Due To
Identifies specimen types and conditions that may cause the specimen to be rejected

Gross hemolysis Reject
Gross lipemia OK
Other Clotted

Specimen Stability Information
Provides a description of the temperatures required to transport a specimen to the performing laboratory, alternate acceptable temperatures are also included

Specimen Type Temperature Time Special Container
Whole Blood EDTA Ambient (preferred) 48 hours PURPLE OR PINK TOP/EDTA

Useful For
Suggests clinical disorders or settings where the test may be helpful

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
Delineates situations when tests are added to the initial order. This includes reflex and additional tests.

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

Clinical Information
Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test

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
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.

References values will be provided in the patient report.

Interpretation
Provides information to assist in interpretation of the test results

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).

Cautions
Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances

This is essentially a qualitative/semiquantitative assay, with the diversity ratio (calculated as described in method description), and visual analysis of the spectratype. This assay does not quantify in any way the amount of transcript for each T-cell receptor. This assay is not a deep sequencing assay and, therefore, does not provide the granularity of information offered by deep sequencing.

 

This assay is intended to generate a spectratype (immunoscope) of the T-cell receptor V beta repertoire and to draw inferences on repertoire diversity based on the number and distribution of peaks across the 24 TCR V beta families.

 

If the CD3+ T-cell count is less than 70 cells/mcL, at least 2 to 3 mL blood may be needed to obtain enough cells to perform the test. For this reason, if only 1 mL blood is provided, this test should not be ordered in patients with completely absent or less than 70 T cells/mcL blood due to underlying disease or treatment, as there may be inadequate sample to amplify TCR V beta families in contexts of such profound T-cell lymphopenia.

 

This test should ideally not be ordered in patients over the age of 40 years without prior discussion with laboratory directors on clinical utility and interpretation in specific clinical contexts.

Clinical Reference
Recommendations for in-depth reading of a clinical nature

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

Special Instructions
Library of PDFs including pertinent information and forms related to the test

Method Description
Describes how the test is performed and provides a method-specific reference

CD3+ T cells are enriched and purified from EDTA whole blood. RNA is obtained from the T cells and converted into cDNA to maximize sample stability. The cDNA is amplified using the polymerase chain reaction (PCR), during which the different CDR3 fragment lengths of the TCR Vb families are fluorescently labeled. The pool of varying CDR3 fragment lengths are separated by size on a capillary electrophoresis genetic analyzer. As the fluorescent label of each CDR3 fragment passes through the laser, the size and fluorescent intensity is recorded. The resulting image is a cluster of fluorescent peaks with single-base-pair separation and different fluorescent intensities, approximately corresponding to the number of fragments of that size represented in the patient’s original RNA. The peak patterns are reviewed for organization (number of peaks), relative intensity across peaks, and size distribution. The number of individual peaks is compared to a reference range established from over 140 healthy donors equally represented by both genders and across the pediatric and adult age spectrum. The reporting units are normalized among the patient population by using a diversity ratio for each TCR V beta family. The diversity ratio for each Vb family is determined by the number of peaks in that specific family relative to all peaks within the patient's sample expressed as a percentage. The analytical process in the laboratory utilizes a variety of controls to assess the performance of the assay and reliability of the result provided. The fragment length analysis is performed by the Gene Marker software and the spectratype is assembled for interpretation. An interpretive report will be provided for each patient sample and includes information on the diversity ratio for each family. The spectratype will be made available on request of the physician.(Unpublished Mayo method)

PDF Report
Indicates whether the report includes an additional document with charts, images or other enriched information

Supplemental

Day(s) Performed
Outlines the days the test is performed. This field reflects the day that the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time before the test is performed. Some tests are listed as continuously performed, which means that assays are performed multiple times during the day.

Varies

Report Available
The interval of time (receipt of sample at Mayo Clinic Laboratories to results available) taking into account standard setup days and weekends. The first day is the time that it typically takes for a result to be available. The last day is the time it might take, accounting for any necessary repeated testing.

20 to 24 days

Specimen Retention Time
Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded

Extracted DNA: 2 months

Performing Laboratory Location
Indicates the location of the laboratory that performs the test

Rochester

Fees
Several factors determine the fee charged to perform a test. Contact your U.S. or International Regional Manager for information about establishing a fee schedule or to learn more about resources to optimize test selection.

  • Authorized users can sign in to Test Prices for detailed fee information.
  • Clients without access to Test Prices can contact Customer Service 24 hours a day, seven days a week.
  • Prospective clients should contact their Regional Manager. For assistance, contact Customer Service.

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 instructions, or as products that do not undergo full FDA review and approval, and are then labeled as an Analyte Specific Reagent (ASR) product.

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 US Food and Drug Administration.

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 Clinic Laboratories interpretation of CPT coding requirements. It is the responsibility of each laboratory to determine correct CPT codes to use for billing.

CPT codes are provided by the performing laboratory.

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)

Test Setup Resources

Setup Files
Test setup information contains test file definition details to support order and result interfacing between Mayo Clinic Laboratories and your Laboratory Information System.

Excel | Pdf

Sample Reports
Normal and Abnormal sample reports are provided as references for report appearance.

Normal Reports | Abnormal Reports

SI Sample Reports
International System (SI) of Unit reports are provided for a limited number of tests. These reports are intended for international account use and are only available through MayoLINK accounts that have been defined to receive them.

SI Normal Reports | SI Abnormal Reports