54 Gel & Capillary | CE-IVD Assays Invivoscribe 2019 | 55 Gel & Capillary CE-IVD Assays IGH + IGK B-Cell Clonality Assays Assay Description The IdentiClone IGH + IGK B-Cell Clonality Assays are in vitro diagnostic products intended for PCR-based detection of clonal immunoglobulin heavy chain and kappa light chain gene rearrangements in patients with suspect lymphoproliferations. Specifically, the IdentiClone IGH + IGK B-Cell Clonality Assays can be used to: • Identify clonality in atypical lymphoproliferative disorders •  Support a differential diagnosis between reactive lesions and hematologic malignancies •  Assign presumptive lineage in mature monoclonal lymphoproliferative disorders •  Identify tumor-specific markers (IGH and IGK gene rearrangements) for post-treatment monitoring • Monitor and evaluate disease recurrence Summary and Explanation of the Test The Invivoscribe CE-marked IdentiClone Assays represent a unique approach to PCR-based clonality testing. These standardized assays were carefully optimized, testing positive and negative control samples using multiplex master mixes. Assay development was followed by extensive validation including the testing of more than 400 clinical samples using Revised European/American Lymphoma (REAL) Classification. Testing was done at more than thirty prominent independent testing centers throughout Europe in a collaborative study known as the BIOMED-2 Concerted Action. Results from this BIOMED-2 study appear in Leukemia, a leading peer-reviewed journal1 . These kits include 6 master mixes to test for rearrangements of both IGH and IGK. The IGH Tube A, B, and C master mixes target the framework 1, 2, 3 regions (respectively) within the variable (VH) region, and the joining (JH) region of the immunoglobulin heavy chain locus. The IGK Tube A master mix targets the variable (Vƙ) and the joining (Jƙ) region. IGK Tube B master mix targets kappa deleting element (Kde) rearrangements with the variable (Vƙ) region and the intragenic Jƙ-Cƙ region. The resulting Vƙ-Kde and Jƙ-Cƙ intron-Kde rearrangements are a result of unsuccessful rearrangements retained by the B cell. For best sensitivity, it is recommended to test suspect B-cell malignancies for both IGH and IGK (van Krieken, JHJM et al., Leukemia. 2007; 21:201 - 206). The included Specimen Control Size Ladder master mix targets multiple genes and generates a series of amplicons of approximately 100, 200, 300, 400, and 600 base pairs to ensure that the quality and quantity of input DNA is adequate to yield a valid result. A single thermocycler program and similar detection methodologies are used with all of our Gene Clonality Assays. This improves consistency and facilitates cross-training on a broad range of different assays. Performance Characteristics Data from two independent studies that tested more than 300 patient samples of varying types suggests the diagnostic accuracy of selected IdentiClone tests to be 96%. In both peer-reviewed studies, there were no clear false-positive results generated using the IdentiClone tests, and there was a high level of precision2 . The clinico-histopathological diagnosis correlated well with PCR results in a higher number of patients when compared with Southern Blot (SB) results, as seen below: PCR/SB concordance1 : IGH: 93% sensitivity / 92% specificity IGK: 90% sensitivity / 90% specificity IGL: 86% sensitivity / 92% specificity TRB: 86% sensitivity / 98% specificity TRG: 89% sensitivity / 94% specificity TRD: 83% sensitivity / 95% specificity PCR vs. SB analysis relative to histopathology and final diagnosis: PCR/SB concordance2 : PCR sensitivity: SB sensitivity: IGH + IGK: 85% 98% 39% TRB: 85% 96% 35% Reference 1.  JJM van Dongen et al., Leukemia 17:2257-2317 (2003). 2.  Y Sandberg et al., J. Mol. Diag. 7(4):495-503 (2005). 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, Intron-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 Gel Electrophoresis Detection Heteroduplex analysis is performed to differentiate clonal and non-clonal PCR products. It involves heat denaturation of double-stranded DNA, followed by a snap chilling process to force DNA strands to quickly reanneal. In non-clonal populations, this process causes the majority of the single-stranded DNA to incorrectly bind to non-homologous strands, resulting in secondary structures and reducing the DNA’s ability to migrate through a non-denaturing polyacrylamide gel. As a result, polyclonal products are frequently observed as smears at high molecular weights. In clonal populations, after heteroduplex analysis is performed, most denatured single-strand PCR products will reanneal correctly with homologous strands of DNA (reforming homoduplexes) allowing them to easily migrate through the polyacrylamide gel as a single band. Therefore, heteroduplex analysis is key for analyzing PCR products visualized using gel detection methods, as it increases the separation between clonal and polyclonal products. Capillary Electrophoresis Detection (ABI) Differential fluorescence detection, such as ABI fluorescence detection, is commonly used to resolve different-sized amplicon products using a capillary electrophoresis instrument. Primers can be conjugated with several different fluorescent dyes (fluorophores), so that they produce different emission spectra upon excitation by a laser in the capillary electrophoresis instrument. In this manner, different fluorescent dyes can correspond to different targeted regions. This detection system results in excellent sensitivity, single nucleotide resolution, differential product detection, and relative quantification. In addition, differential detection allows accurate, reproducible and objective interpretation of primer-specific products. Inter- assay and intra-assay reproducibility in size determination using capillary electrophoresis is approximately 1 to 4 nucleotides. Ordering Information Catalog # Products Quantity 9-100-0010 IdentiClone™ IGH + IGK B-Cell Clonality Assay - Gel Detection 33 reactions 9-100-0020 IdentiClone™ IGH + IGK B-Cell Clonality Assay MegaKit - Gel Detection 330 reactions 9-100-0031 IdentiClone™ IGH + IGK B-Cell Clonality Assay - ABI Fluorescence Detection 33 reactions 9-100-0041 IdentiClone™ IGH + IGK B-Cell Clonality Assay MegaKit - ABI Fluorescence Detection 330 reactions These are in vitro diagnostic products, and are not available for sale or use within North America. This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936. Figure Legend: Simple representation of the organization of a rearranged immunoglobulin heavy chain (IGH) gene on chromosome 14q32.33 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 κ