66 Gel & Capillary | CE-IVD Assays Invivoscribe 2019 | 67 Gel & Capillary CE-IVD Assays T-Cell Receptor Gamma Gene Rearrangement Assay 2.0 Assay Description 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 suspected lymphoproliferations. Summary and Explanation of the Test 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 loci1 . The Invivoscribe designed CE-marked IdentiClone Assays represents an improved approach to PCR-based clonality testing of lymphoproliferative disorders, as it can detect the vast majority of T-cell receptor gamma (TRG, formerly known as TCRG) gene rearrangements with a single multiplex master mix. This assay allows for amplification of the TRG region with fluorescent labeled primers, yielding products that can be grouped under a single Gaussian distribution when separated by size using capillary electrophoresis. In addition, the product size facilitates increased success when testing FFPE samples. The included analysis algorithm aids in the interpretation of data and identification of significant clonal peaks. Presence or absence of molecular clonality can support the differential diagnosis of reactive lesions and certain B- and T-cell malignancies, provided that the results are interpreted in the context of all available clinical, histological, and immunophenotypic data. Performance Characteristics To assess the performance of the TCRG 2.0 Assay, testing was performed on cell lines with known clonal rearrangements followed by testing on previously sequenced clinical samples. When used in combination with the provided TCRG Algorithm worksheet, the assay was capable of detecting DNA from 6 control cell lines (200 ng/µL) diluted into polyclonal tonsil DNA (200 ng/µL) at 5% (v/v). Furthermore, the performance of the TCRG 2.0 Assay was evaluated on clinical samples for which the T-cell receptor gamma gene rearrangement status had been identified by Roche 454 sequencing. For the 7 samples that had been identified as clonal by sequencing, the TCRG 2.0 assay had 100% concordance. For the 12 samples that were either negative for a clonal event or were oligoclonal, concordance of the TCRG 2.0 assay was 75%. Sample types included peripheral blood, bone marrow, and formalin-fixed, paraffin embedded (FFPE) tissue. The results of molecular clonality tests should always be interpreted in the context of clinical, histological and immunophenotypic data. Reference 1.  Miller JE, Wilson SS, Jaye DJ, and Kronenberg M. Mol. Diag. 1999, 4(2):101-117. Reagents Controls Concentration Units in Assay Units in Assay MegaKit 5% TCRG Positive Control DNA 50 μg/mL 1 x 50 μL tube 5 x 50 μL tube TCRG Negative Control DNA 50 μg/mL 1 x 50 μL tube 5 x 50 μL tube Master Mixes Target Units in Assay Units in Assay MegaKit TCRG - 6FAM Vƴ 1-Vƴ 11 + Jƴ 1/Jƴ 2, Jƴ P, Jƴ P1/Jƴ P2 1 x 1500 μL tube 10 x 1500 μL tubes Specimen Control Size Ladder Multiple Genes 1 x 1500 μL tube 10 x 1500 μL tubes Ordering Information Catalog # Products Quantity 9-207-0101 IdentiClone™ T-Cell Receptor Gamma Gene Rearrangement Assay 2.0 - ABI Fluorescence Detection 33 reactions 9-207-0111 IdentiClone™ T-Cell Receptor Gamma Gene Rearrangement Assay MegaKit 2.0 - ABI Fluorescence Detection 330 reactions Capillary Electrophoresis Detection (ABI) Fluorescence detection is commonly used to resolve the different sized amplicon products using a capillary electrophoresis instrument. Primers are conjugated with a 6-FAM fluorescent dye (fluorophore), so that they can be detected upon excitation by laser. This detection system results in a high sensitivity, single nucleotide size resolution, and relative quantification. Inter- and intra-assay reproducibility in size determination using capillary electrophoresis is approximately 1 to 4 nucleotides. The data shown were generated using the TCRG–6FAM master mix. Amplified products were run on an ABI 3130 instrument. These are in vitro diagnostic products, and are not available for sale or use within North America. Figure Legend: Simple representation of the organization of the T-cell receptor gamma gene on chromosome 7p14. Black arrows represent the relative positions of primers that target the variable region genes and the downstream joining region gene segments that are involved in rearrangements in T-cell lymphomas. The downstream primers are fluorescently labeled through the incorporation of a 6FAM fluorophore. The amplicon products generated from these rearrangements are detected by capillary electrophoresis. This assay was developed by Invivoscribe. The performance of this assay was reviewed and validated by the EuroClonality/BIOMED-2 Group. Euroclonality manuscript in preparation: multicenter study with 250 clinical patient specimens.