94 Gel & Capillary | RUO Assays Invivoscribe 2019 | 95 Gel & Capillary RUO Assays IGK Gene Clonality Assays Assay Use IGK 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 Two master mixes target conserved regions within the variable (Vƙ1-7) and the joining (Jƙ1-5) regions that flank the unique hypervariable, antigen-binding, complementarity determining region 3 (CDR3). Other primers target the Kde and intragenic regions. Tube A contains six upstream primers and two Jƙ region primers. Tube B contains six upstream Vƙ region primers, an upstream intragenic primer and a downstream Kde primer. 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 immunoglobulin kappa (IGK) light chain locus on the short arm of chromosome 2 (2p12, formerly 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 lymphocytes1 . 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 complementarity determining region 3 (CDR3) in kappa light chain genes is limited and rearrangements in this region display significant skewing (platykurtosis)2 . Therefore, clonal CDR3 products generated from this region are most easily and reliably identified by heteroduplex analysis using standard polyacrylamide gels. Alternatively, capillary electrophoresis or gene sequencing instruments coupled with differential fluorescence detection can be used for analysis. 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. AW Langerak et al., Leukemia 17: 2275-2280 (2003). 2.  EP Rock, PR Sibbald, MM Davis, and YH Chien. J. Exp. Med. 179(1): 323-328 (1994). 3. JJM van Dongen et al., Leukemia 17: 2257-2317 (2003). Reagents Ordering Information Catalog # Products Quantity 1-102-0020 IGK Gene Clonality Assay - Gel Detection 33 reactions 1-102-0030 IGK Gene Clonality Assay MegaKit - Gel Detection 330 reactions 1-102-0021 IGK Gene Clonality Assay - ABI Fluorescence Detection 33 reactions 1-102-0031 IGK 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 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-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 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 Figure Legend: Schematic diagram of the immunoglobulin kappa light chain gene complex on chromosome 2p11.2. Shown are the relative positions and orientations for the Vƙ-Jƙ, and Kde primers, which are included in the IGK master mix tubes. This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.