108 Gel & Capillary | RUO Assays Invivoscribe 2019 | 109 Gel & Capillary RUO Assays TCRD Gene Clonality Assays Assay Use TCRD Gene Clonality Assays are useful for studies involving: •  Identification of clonal T-cell populations highly suggestive of T-cell malignancies • Lineage determination of leukemias and lymphomas • Monitoring and evaluation of disease recurrence • Detection and assessment of residual disease • Evaluation of new research and methods in malignancy studies Summary and Explanation of the Test The TCRD Tube Master Mix targets conserved regions within the variable (Vδ1-6), the diversity (Dδ2-3) and the joining (Jδ1-4) regions that flank the unique, hypervariable, antigen-binding, complementarity determining region 3 (CDR3) of the T-cell receptor delta (TRD, formerly known as TCRD). Positive and negative controls, as well as a Specimen Control Size Ladder Master Mix, are included. PCR products can be analyzed by capillary electrophoresis or heteroduplex analysis. Clonality is indicated if the master mix generates clonal products. Background The human T-cell receptor delta (TRD, formerly known as TCRD) gene locus is comprised of a cluster of 10 genes located on chromosome 14 (14q11.2) spread over 60 kilobases, localized between the T-cell receptor alpha (TRA, formerly known as TCRA) variable (Vα) and joining (Jα) gene segments. It is made up of 8 variable (Vδ), 3 diversity (Dδ), and 4 joining (Jδ) gene segments1 . At least 5 of the 8 Vδ gene segments can also rearrange to Jδ gene segments and other Vδ gene segments may also be utilized in TRD gene rearrangements in rare cases. Although the small number of Vδ, Dδ, and Jδ gene segments available for recombination limits the potential combinatorial diversity, the complementarity determining region 3 (CDR3) or junctional diversity is extensive due to the addition of N regions, P regions, and random deletion of nucleotides by recombinases. This diversity is also extended by the recombination of up to three Dδ segments and therefore up to four N regions within the rearranged TRD locus. This limited germline diversity encoded at the TRD locus in conjunction with extensive junctional diversity results in a useful target for PCR analysis. TRD recombination events have been used most extensively as clonal markers in both T- and B-cell ALL. This standardized multiplex PCR assay detects the vast majority of clonal TRD gene rearrangements using a single multiplex master mix1 . This assay provides rapid TCR clonality assessment, reducing or eliminating the number of Southern blot tests performed in the laboratory2 . The detection rate of clonal TRD gene rearrangements using this assay is exceptionally high1 . The performance characteristics of this assay have been independently determined by the EuroClonality/BIOMED-2 Group. This consortium of 47 molecular diagnostic laboratories tested hundreds of clinical samples, taking into account their lymphoproliferative status as defined by the WHO classifications2 . Specimen Requirements 1.  5 mL of peripheral blood, bone marrow biopsy, or bone marrow aspirate anti-coagulated with heparin or EDTA; or, 2. Minimum 5 mm cube of tissue; or, 3. 2 μg of genomic DNA; or, 4. Formalin-fixed, paraffin-embedded (FFPE) tissue or slides. Reference 1. FL Lavender et al., Leukemia 17: 2292-2296 (2003). 2. JJM van Dongen et al., Leukemia 17: 2257-2317 (2003). Reagents Ordering Information Catalog # Products Quantity 1-206-0010 TCRD Gene Clonality Assay - Gel Detection 33 reactions 1-206-0020 TCRD Gene Clonality Assay MegaKit - Gel Detection 330 reactions 1-206-0011 TCRD Gene Clonality Assay - ABI Fluorescence Detection 33 reactions 1-206-0021 TCRD Gene Clonality Assay MegaKit - ABI Fluorescence Detection 330 reactions Gel Electrophoresis Detection The data shown was generated using the master mix indicated. Amplified products were heteroduplexed and run on a 6% non-denaturing polyacrylamide/TBE gel. Lane 1 is data generated testing an alternative 100% clonal control DNA; lane 2 is data generated testing the recommended 100% clonal control DNA; lane 3 is data generated testing a 10% dilution of the recommended clonal control DNA; and, lane 4 is data generated testing IVS-0000 Polyclonal Control DNA (Cat# 4-092- 0010). The IVS-0000 control is used as a negative control for many of our tests. A standard 100 base pair DNA size ladder was run in the lanes flanking the test samples. Capillary Electrophoresis Detection (ABI) The data shown was generated using the master mix indicated. Amplified products were run on an ABI 3100 instrument. Panel 1 displays data generated testing an alternative 100% clonal control DNA; panel 2 displays data generated testing the recommended 100% clonal control DNA; panel 3 displays data generated testing a 10% dilution of the recommended clonal control DNA; and, panel 4 displays data generated testing the IVS-0000 Polyclonal Control DNA (Cat# 4-092-0010). Our IVS-0000 DNA is often tested to provide information regarding valid size ranges for each master mix. Controls Concentration Units in Assay Units in Assay MegaKit IVS-0021 Clonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-0000 Polyclonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes Master Mixes Target Units in Assay Units in Assay MegaKit TCRD Tube Multiple Vδ + Dδ + Jδ 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 Figure Legend: Simplified diagram of a representative rearranged T-cell receptor delta gene showing the approximate placement of the upstream and downstream DNA primers. The numbers of primers and their specificity are listed for the TRD Tube Master Mix tube. This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.