88 Gel & Capillary | RUO Assays Invivoscribe 2019 | 89 Gel & Capillary RUO Assays Assay Use IGH + IGK B-Cell 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 PCR master mixes are included in these test kits to test for rearrangements of both IGH and IGK. IGH Tubes A, B, and C target the conserved framework 1, 2, and 3 regions (respectively) within the variable (VH) region and the joining (JH) region of the IGH locus. IGK tubes A and B target the variable (Vƙ), intragenic and joining (Jƙ), and kappa deleting element (Kde) regions of the IGK locus. Positive and negative controls, as well as 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 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 VH3, VH4, and VH1 families, which together cover 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 . The human immunoglobulin kappa (IGK) light chain locus on the short arm of chromosome 2 (2p11.2) spans 1820 kb. It is made up of 76 variable (Vƙ) gene segments belonging to 7 subgroups, 5 joining (Jƙ) gene segments, and one constant (Cƙ) gene segment. Productive assembly of the kappa gene is successful in about 60% of human B lymphocytes2 ; however, even when unsuccessful, clonal B cells generally retain the rearranged kappa genes. The Vƙ segments encode the first 95 N-terminal amino acids. Positions 96-108 are encoded by one of five joining (Jƙ) gene segments. The constant (Cƙ) portion of the kappa light chain (amino acids 109-214) is encoded by a single constant (Cƙ) region separated from the Jƙ region by an intron. The length of the hypervariable CDR3 in kappa light chain genes is limited and rearrangements in this region display significant skewing (platykurtosis)3 . Therefore, clonal CDR3 products generated from this region 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 classifications4 . 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.  EP Rock, PR Sibbald, MM Davis, and YH Chien. J. Exp. Med. 179(1): 323-328 (1994). 4. JJM van Dongen et al., Leukemia 17: 2257-2317 (2003). Reagents 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-0007 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 IGK Tube A Vƙ-Jƙ 1 x 1500 μL tube 10 x 1500 μL tubes IGK Tube B Vƙ-Kde 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 1-100-0010 IGH + IGK B-Cell Clonality Assay - Gel Detection 33 reactions 1-100-0020 IGH + IGK B-Cell Clonality Assay MegaKit - Gel Detection 330 reactions 1-100-0031 IGH + IGK B-Cell Clonality Assay - ABI Fluorescence Detection 33 reactions 1-100-0041 IGH + IGK B-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 then run on a 6% non-denaturing polyacrylamide/TBE gel. Lane 1 is data generated testing an alternative 100% clonal control DNA; lane 2 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). This DNA 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. Figure Legend: Simple representation of the organization of a rearranged immunoglobulin heavy chain (IGH) gene on chromosome 14 and the immunoglobulin kappa light chain gene on chromosome 2p11.2. Black arrows represent the relative positions of primers that target the conserved framework regions (FR1-3) and the downstream consensus JH gene segments for IGH and the Vƙ, Jƙ, INTR and Kde primers which are included in the IGK master mix tubes. κ IGH + IGK B-Cell Clonality Assays This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.