98 Gel & Capillary | RUO Assays Invivoscribe 2019 | 99 Gel & Capillary RUO Assays TCRB + TCRG T-Cell Clonality Assays Assay Use TCRB + TCRG T-Cell 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 Five master mixes are included in these test kits to test for rearrangements of both TRB (formerly known as TCRB) and TRG (formerly known as TCRG). TCRB Tubes A and B target framework regions within the variable region, and the joining region of the T-cell receptor beta locus. TCRB Tube C targets the diversity and joining regions. TCRG Tubes A and B target framework regions within the variable region, and the joining region of the T-cell receptor gamma locus. 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 any one of the master mixes generates clonal products. Background The human T-cell receptor beta (TRB, formerly known as TCRB) gene locus on chromosome 7 (7q34, formerly 7q35) includes 64-67 variable (Vβ) gene segments (belonging to 30 subgroups), 2 diversity (Dβ) gene segments, and 13 joining (Jβ) gene segments, spread over 685 kilobases. The diversity of this locus has complicated PCR-based testing, however, this standardized multiplex PCR assay detects the vast majority of clonal TRB gene rearrangements using only three multiplex master mixes. The detection rate of clonal TRB gene rearrangements using this assay is exceptionally high1 . The T-cell receptor gamma (TRG, formerly known as TCRG) chain locus spans 128 kb on chromosome 7 (7p14). Rearrangement of the variable (Vƴ ) and joining (Jƴ ) genetic segments of the TRG locus result in Vƴ-Jƴ products of unique length and sequence. The TRG locus does not contain D segments2 . In addition, the TRG gene contains a limited number of Vƴ and Jƴ segments such that the amplification of all major Vƴ-Jƴ combinations is possible with four Vƴ and two Jƴ primers. This standardized multiplex PCR assay detects the vast majority of clonal TRG gene rearrangements using two multiplex master mixes3 . Using this assay, the clonal TRG gene rearrangement detection rate is exceptionally high3 . PCR products generated from the TRB and TRG assays are easily and reliably identified by heteroduplex analysis or capillary electrophoresis. 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. M Brüggemann et al., Leukemia 17: 2283-2289 (2003). 2. JJM van Dongen et al., Leukemia 17: 2257-2317 (2003). 3. K Beldjord et al., Leukemia 17: 2289-2292 (2003). Reagents Ordering Information Catalog # Products Quantity 1-200-0010 TCRB + TCRG T-Cell Clonality Assay - Gel Detection 33 reactions 1-200-0020 TCRB + TCRG T-Cell Clonality Assay MegaKit - Gel Detection 330 reactions 1-200-0011 TCRB + TCRG T-Cell Clonality Assay - ABI Fluorescence Detection 33 reactions 1-200-0021 TCRB + TCRG T-Cell 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 the master mix. Figure Legend: Simplified diagram of a representative rearranged T-cell receptor beta gene and the T-cell receptor gamma gene showing the approximate placement of the upstream and downstream DNA primers. The numbers of primers and their specificity are listed for master mix TCRB Tubes A, B, and C and TCRG Tubes A and B. (The Vƴ1f primer is a consensus primer that targets Vƴ 1 through Vƴ 8). Controls Concentration Units in Assay Units in Assay MegaKit IVS-0009 Clonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-0004 Clonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes 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 TCRB Tube A Multiple Vβ + Jβ1/2 1 x 1500 μL tube 10 x 1500 μL tubes TCRB Tube B Multiple Vβ + Jβ2 1 x 1500 μL tube 10 x 1500 μL tubes TCRB Tube C Multiple Dβ + Jβ1/2 1 x 1500 μL tube 10 x 1500 μL tubes TCRG Tube A Vƴ 1-8 + Vƴ 10 + Jƴ 1 x 1500 μL tube 10 x 1500 μL tubes TCRG Tube B Vƴ 9+ Vƴ 11 + Jƴ 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 This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.