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Test Code MISC2MAYOTCRVB 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

Method Name

Molecular TCR Vb-CDR3 Fragment Length Analysis

Reporting Name

TCR Spectratyping, B

Specimen Type

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

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


-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

Reject Due To

Gross hemolysis Reject
Gross lipemia OK
Other Clotted

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.


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).

Method Description

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)

Day(s) Performed


Report Available

20 to 24 days

Performing Laboratory

Mayo Clinic Laboratories in Rochester

Test 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 US 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


TCR V beta Spectratyping Assay Patient Information (T719) in Special Instructions

Secondary ID