92 Gel & Capillary | RUO Assays Invivoscribe 2019 | 93 Gel & Capillary RUO Assays IGH Gene Clonality Assays Assay Use IGH Gene Clonality Assays are useful for studies involving: •  Identification of clonal B-cell populations highly suggestive of B-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 target conserved regions within the variable (VH), diversity (DH), and the joining (JH) regions that flank the unique hypervariable, antigen-binding, complementarity determining region 3 (CDR3). Tube A contains six framework region 1 (FR1) primers and a consensus JH region primer. Tube B contains seven framework region 2 (FR2) primers and a consensus JH primer. Tube C contains seven framework region 3 (FR1) primers and a consensus JH primer. Tube D contains six DH region primers and a consensus JH region primer. Tube E contains a DH7 region primer and a consensus JH primer. Positive and negative controls, as well as the 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 a clonal product. Background The immunoglobulin heavy chain (IGH) gene locus on chromosome 14 (14q32.33, formerly 14q32.3) includes 46-52 functional and 30 nonfunctional variable (VH), 27 functional diversity (DH), and 6 functional joining (JH) gene segments spread over 1250 kilobases1,2 . The most frequently used VH gene segments in normal and malignant B cells belong to the VH3, VH4, and VH1 family, together covering 75–95% of VH usage. The VH gene segments contain three framework regions (FR) and two complementarity determining regions (CDR). The FRs are characterized by their similarity among the various VH segments, whereas the CDRs are highly different even within the same VH family. The CDRs represent the preferred target sequences for somatic hypermutations; however, somatic mutations can also occur in the FRs. Therefore, family-specific primers in the three different FRs were designed to increase the detection rate of clonal IGH B-cell populations and decrease the occurrence of false-negative results due to somatic hypermutation in primer binding sites1 . In addition to VH-JH rearrangements, incomplete DH-JH rearrangements have been found in mature and immature B-cell malignancies. Therefore, DH-JH PCR analysis may be of added value for clonality assessment2 . 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 classifications3 . 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 Hummel et al., Leukemia 17:2266-2272 (2003). 2. AW Langerak et al., Leukemia 17:2272-2275 (2003). 3. JJM van Dongen et al., Leukemia 17:2257-2317 (2003). Reagents Ordering Information Catalog # Products Quantity 1-101-0020 IGH Gene Clonality Assay - Gel Detection 33 reactions 1-101-0040 IGH Gene Clonality Assay MegaKit - Gel Detection 330 reactions 1-101-0061 IGH Gene Clonality Assay - ABI Fluorescence Detection 33 reactions 1-101-0081 IGH 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 then run on a 6% non-denaturing polyacrylamide/TBE gel. Lane 1 is data generated testing an alternative 100% clonal control DNA; lane 2 data is 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). IVS-0000 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. For the master mix: 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-0030 Clonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-0019 Clonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-0024 Clonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-0008 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 IGH Tube A Framework 1 + JH 1 x 1500 μL tube 10 x 1500 μL tubes IGH Tube B Framework 2 + JH 1 x 1500 μL tube 10 x 1500 μL tubes IGH Tube C Framework 3 + JH 1 x 1500 μL tube 10 x 1500 μL tubes IGH Tube D DH1-6 + JH 1 x 1500 μL tube 10 x 1500 μL tubes IGH Tube E DH7 + JH 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: Simple representation of the organization of a rearranged immunoglobulin heavy chain gene on chromosome 14. Black arrows represent the relative positions of primers that target the conserved framework (FR1- 3) and diversity (DH1-7) regions, and the downstream consensus JH gene segments. The amplicon products generated from each of these regions can be differentially detected when fluorescent primer sets are used with capillary electrophoresis instruments that employ differential fluorescence detection. This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.