96 Gel & Capillary | RUO Assays Invivoscribe 2019 | 97 Gel & Capillary RUO Assays IGL Gene Clonality Assays Assay Use IGL 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 The IGL Tube master mix targets conserved regions within the variable (Vʎ1-3) and the joining (Jʎ1-3) regions that flank the unique, hypervariable, antigen-binding, complementarity determining region 3 (CDR3). 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 immunoglobulin lambda (IGL) light chain locus is located on the long arm of chromosome 22 (22q11.2) and spans 1050 kilobases. It is made up of 73-74 variable (Vʎ) gene segments (spread over 900 kilobases), 7-11 joining (Jʎ) gene segments and 7-11 constant (Cʎ) gene segments depending on the haplotypes. Of the 73-74 Vʎ region genes, only 30-33 are functional and can be grouped into 11 families and 3 clans1 . The Jʎ and Cʎ region genes are organized in tandem with a Jʎ segment preceding a Cʎ gene. Typically there are 7 Jʎ-Cʎ segments of which 4 are functional and encode the 4 Ig lambda isotypes. IGL gene rearrangements are present in 5-10% of Ig kappa B-cell malignancies and in all Ig lambda B-cell malignancies. Therefore, Vʎ-Jʎ rearrangements potentially represent an attractive extra PCR target for clonality studies to compensate for false-negative IGH VH-JH PCR results mainly caused by somatic hypermutations. It should be noted that because of the limited size of the junctional region, it is extremely difficult to distinguish polyclonal from monoclonal rearrangements by running a simple agarose or polyacrylamide gel1 . Therefore, clonal Vʎ-Jʎ PCR products 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 analysis1 . 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. F Davi et al., Leukemia 17:2280-2283 (2003). 2. JJM van Dongen et al., Leukemia 17:2257-2317 (2003). Reagents Ordering Information Catalog # Products Quantity 1-103-0010 IGL Gene Clonality Assay - Gel Detection 33 reactions 1-103-0020 IGL Gene Clonality Assay MegaKit - Gel Detection 330 reactions 1-103-0011 IGL Gene Clonality Assay - ABI Fluorescence Detection 33 reactions 1-103-0021 IGL 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-000 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-0010 Clonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-0029 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 IGL Tube Vʎ-Jʎ 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 lambda light chain gene complex on chromosome 22q11.2. Shown are the relative positions and orientations for the Vʎ and Jʎ primers, which are included in the IGL master mix tube. The two Vʎ primers only target Vʎ1, 2, and 3 because these three V families cover approximately 70% of rearrangeable Vʎ gene segments, and approximately 90% of all IGL gene rearrangements involve these three families. Similarly, the single Jʎ primer only targets Jʎ1, 2, and 3 because these three J segments are involved in 98% of all IGL gene rearrangements. This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.