CAT #: 92070111
IdentiClone® T-cell Receptor Gamma Gene Rearrangement Assay 2.0 MegaKit - ABI Fluorescence Detection
The IdentiClone T-Cell Receptor Gamma Gene Rearrangement Assay 2.0 is an in vitro diagnostic product intended for PCR-based detection of clonal T-cell receptor gamma chain gene rearrangements in patients with suspect lymphoproliferations.
Specifically, the T-Cell Receptor Gamma Gene Rearrangement Assay 2.0 can be used to identify clonality in suspect lymphoproliferations.
Summary and Explanation of the Test
Rearrangements of the antigen receptor genes occur during ontogeny in B and T lymphocytes and generate products that are unique in length and sequence. Polymerase chain reaction (PCR) assays can be used to identify lymphocyte populations derived from a single cell by detecting the unique V-J gene rearrangements present within these antigen receptor loci.1 This IdentiClone PCR assay employs multiple consensus DNA primers that target conserved genetic regions within the T cell receptor gamma chain gene and amplify the region with fluorescently labeled primers, followed by fractionation by capillary electrophoresis and analysis by instrument software. This DNA based test is used to detect the vast majority of clonal T-cell populations. Presence or absence of clonality can support the differential diagnosis of reactive lesions and certain T- and B-cell malignancies.
This assay cannot reliably detect clonality present at less than 5% of the total lymphocyte population. Always interpret the results of molecular clonality testing in the context of all available clinical, histological and immunophenotypic data.
This test kit consists of a single master mix that contains primers that target the Vγ2, 3, 4, 5, 8, 9, 10, and 11 and Jγ1/Jγ2, JγP, and JγP1/JγP2 regions, generating PCR amplicons with an expected size range between 159 and 207 nucleotides (nt). The Specimen Control Size Ladder master mix targets multiple genes and generates a series of amplicons of approximately 100, 200, 300, 400, and 600 nucleotides to ensure that the quality and quantity of input DNA is adequate to yield a valid result. The procedure uses a single thermal cycler program and similar detection methodology for all Invivoscribe Gene Clonality Assays, which improves consistency and facilitates cross training on a broad range of assays.
Peak analysis is supported by a software based algorithm that calculates the relative peak height ratio (RPR) and a statistical parameter D(x) value for each peak. The RPR is calculated by dividing the height of each peak to the smaller of its neighboring peaks and it must exceed a cutoff of 4.0. The D(x) value is based on a variation of the Kolmogorov-Smirnov test, which compares two empirical distributions and determines whether they are statistically different and its value must be greater than 0.0419.
Principles of the Procedure
Polymerase Chain Reaction (PCR)
PCR assays are routinely used for the identification of clonal T-cell populations. This test amplifies the DNA between primers that target conserved regions within the variable (V) and the joining (J) regions that flank the unique hypervariable antigen-binding region 3 (CDR3). These conserved regions lie on either side of an area within the V-J region where programmed genetic rearrangements occur during maturation of all B and T lymphocytes. The antigen receptor genes that undergo rearrangement are the immunoglobulin heavy chain and light chains in B-cells, and the T-cell receptor genes in T-cells. Each B- and T-cell has a single productive V-J rearrangement that is unique in both length and sequence. Therefore, when DNA from a normal or polyclonal population is amplified using primers that flank the V-J region, a Gaussian distribution (bell-shaped curve) of amplicon products within an expected size range is generated. This Gaussian distribution reflects the heterogeneous population of V-J rearrangements. (In certain cases, where lymphocyte DNA is not present, no product is detected.) DNA from samples containing a clonal population yield one or two prominent amplified products (amplicons) within a diminished polyclonal background.
Since the antigen receptor genes are polymorphic (consisting of a heterogeneous population of related DNA sequences), it is difficult to employ a single set of DNA primer sequences to target all of the conserved flanking regions around the V-J rearrangement. N-region diversity, and somatic mutation further scramble the DNA sequences in these regions. Therefore, a multiplex master mix, which targets multiple V and J regions, is required to detect the majority of clonal rearrangements. As indicated, clonal rearrangements are identified as one or two prominent, single-sized products within the background of different-sized amplicon products that form the Gaussian distribution around a statistically favored, average-sized rearrangement.
Fluorescence detection is commonly used to resolve the different-sized amplicon products using a capillary electrophoresis instrument. Primers are conjugated with a 6FAM fluorescent dye (fluorophore) so that they can be detected after excitation by a laser in the capillary electrophoresis instrument. This highly sensitive detection system provides single nucleotide size resolution and relative quantification. Inter and intra-assay reproducibility in size determination using capillary electrophoresis is approximately 1 to 2 nucleotides. This reproducibility and sensitivity coupled with the automatic archiving of specimen data allows for the monitoring, tracking, and comparison of data from individual patients over time.
This assay tests genomic DNA extracted and purified from peripheral blood, bone marrow aspirates or paraffin embedded tissue.
1. Miller, JE et al. (1999) Molecular Diagnostics. 4(2):101-117.
This assay was developed by Invivoscribe. The performance of this assay was reviewed and validated by the EuroClonality/BIOMED-2 Group.
Warranty and Liability
Invivoscribe, Inc. (Invivoscribe®) is committed to providing the highest quality products. Invivoscribe® warrants that the products meet or exceed the performance standards described in the Instructions For Use, as to products with such an insert. If a product is covered by product specifications and does not perform as specified, our policy is to replace the product or credit the full purchase price. No other warranties of any kind, expressed or implied, are provided by Invivoscribe®. Invivoscribe® liability shall not exceed the purchase price of the product. Invivoscribe® shall have no liability for direct, indirect, consequential or incidental damages arising from the use, results of use, or inability to use its products; product efficacy under purchaser controlled conditions in purchaser’s laboratory must be established and continually monitored through purchaser defined and controlled processes including but not limited to testing of positive, negative, and blank controls every time a sample is tested. Ordering, acceptance, and use of product constitutes purchaser acceptance of sole responsibility for assuring product efficacy and purchaser agreement to the limitation of liability set forth in this paragraph.
This product is an in vitro diagnostic product is not available for sale or use within North America.
This product is covered by one or more of the following: European Patent Number 1549764, European Patent Number 2418287, European Patent Number 2460889, Japanese Patent Number 4708029, United States Patent 8859748, United States Patent 10280462, and related pending and future applications. All of these patents and applications are licensed exclusively to Invivoscribe®. Additional patents licensed to Invivoscribe® covering some of these products apply elsewhere. Many of these products require nucleic acid amplification methods such as Polymerase Chain Reaction (PCR). No license under these patents to use amplification processes or enzymes is conveyed expressly or by implication to the purchaser by the purchase of this product.
IdentiClone® is a registered trademark of Invivoscribe®.
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