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 rearrangement in patients with suspect lymphoproliferations.

Specifically, the T Cell Receptor Gamma Gene Rearrangement Assay 2.0 can be used to identify clonality in patients with lymphoproliferations.

IdentiClone™ assay kits are in vitro diagnostic products. These standardized PCR-based tests come with a Standard Operating Procedure (SOP), an interpretative guide, master mixes, and controls. Master mixes are composed of a buffered magnesium solution, deoxynucleotides, and multiple primers that target the gene segments of interest. Multiple primers are used to ensure a more comprehensive testing approach necessary to reliably identify clonal rearrangements. These assay kits are complete with the exception of Taq DNA polymerase, which is not provided. A single thermocycler program and similar detection methods are used within each series of kits to improve consistency, reduce human error, and facilitate cross training.

Standard Protocol:

1. Using gloved hands, remove the master mixes from the freezer. Allow the tubes to thaw; then gently vortex to mix.
2. In a containment hood or dead air box remove an appropriate aliquot to clean, sterile microfuge tube (one tube for each of the master mixes). Aliquot volumes should be 45ul for each sample + 135ul for the positive, negative and no template controls. We recommend adding an additional 20ul to correct for pipetting errors.
3. Add the appropriate amount of either AmpliTaq Gold or AmpliTaq DNA polymerase (0.25ul of either AmpliTaq Gold or AmpliTaq @ 5U/ul per 50ul total PCR reaction volume) to each of the master mixes and gently mix by inverting several times or gently vortexing.
4. Aliquot 45ul of master mix to individual wells of a PCR plate.
5. Add 5ul of DNA from the unknown and control samples to individual tubes or wells containing the respective master mix reactions, and pipette up and down several times to mix. Amplify target DNA using the universal thermocycler program.

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Rearrangements of the antigen receptor genes occur during ontogeny in B and T lymphocytes. These gene rearrangements 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. This IdentiClone™ PCR assay employs multiple consensus DNA primers that target conserved genetic regions within the T cell receptor gamma chain gene. Amplifying the region with fluorescently labeled primers is 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. It should be emphasized that the results of molecular clonality testing should always be interpreted 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 Vg2, 3, 4, 5, 8, 9, 10, & 11 and Jg1/Jg2, JgP, and JgP1/JgP2 regions. The PCR amplicons have an expected size range between 159 and 207 base pairs. The Specimen Control Size Ladder master mix targets multiple genes and generates a series of amplicons of approximately 100, 200, 300, 400, and 600 base pairs to ensure that the quality and quantity of input DNA is adequate to yield a valid result. A single thermocycler program and similar detection methodology is used with all of our Gene Clonality Assays. This improves consistency and facilitates cross training on a broad range of our different assays.

A software based algorithm has been developed for analyzing the peaks that were measured on the capillary electrophoresis instrument. The algorithm 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.

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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 DNA primers that flank the V-J region, a Gaussian distribution (bell-shaped curve) of amplicon products is produced within an expected size range. This Gaussian distribution reflects the heterogeneous population of V-J rearrangements. In certain cases, where lymphocyte DNA is not present, no product is detected. For DNA from samples containing a clonal population, the yield is one or two prominent amplified products (amplicons) within the valid size range.

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 (Figure 1), 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.

Differential Fluorescence Detection
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 base pair size resolution and relative quantification. Inter and intra-assay reproducibility in size determination using capillary electrophoresis is approximately 1 to 2 base pairs. 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.

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This assay tests genomic DNA extracted and purified from peripheral blood, bone marrow aspirates or paraffin embedded tissue.

This is an in vitro diagnostic product and is not available for sale or use within the United States. This product is covered by issued United States Patent Number 7,785,783 and other pending patent applications originating from International Patent Application PCT/AU2004/000625, European Patent Application Number 04732551.9 (16 countries), Brazil Patent Application Number PI0410283.5, Canadian Patent Application Number 2525122, Indian Patent Application Number 5792/DELNP/2005, Japanese Patent Application Number 2006-529437, Mexican Patent Application Number PA/a/2005/012102, Chinese Patent Application Number 200480016603.5, and Korean Patent Application Number 10-2005-7021561, all of which are licensed exclusively to Invivoscribe Technologies, Inc.

Use of this product may require nucleic acid amplification methods such as Polymerase Chain Reaction (PCR). No patent license to use amplification processes or enzymes is conveyed expressly or by implication to the purchaser by the purchase of these products.

Purchase of this product includes a limited sublicense for non-commercial uncompensated practice of this technology if and only if the purchaser is registered with IVS as an exclusively non-commercial user of IVS products. No sublicense for such use is granted simply by purchase of these products. To request a form for registration as an exclusively non-commercial product user, to discuss terms for a potential sublicense for broader practice of these methods, or for other questions please contact IVS by email at legal@invivoscribe.com, or by telephone at +1 858 224 6600.

IdentiClone™ is a trademark of Invivoscribe Technologies, Inc.

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