64 Gel & Capillary | CE-IVD Assays Invivoscribe 2019 | 65 Gel & Capillary CE-IVD Assays TCRB Gene Clonality Assays Assay Description The IdentiClone TCRB Gene Clonality Assays are in vitro diagnostic products intended for PCR-based detection of clonal T-cell receptor beta chain gene rearrangements in patients with suspect lymphoproliferations. Specifically, the IdentiClone TCRB Gene Clonality Assay can be used to: • Identify clonality in suspected lymphoproliferations •  Support a differential diagnosis between reactive lesions and T-cell and some immature B-cell malignancies •  Determine lineage involvement in mature lymphoproliferative disorders • 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 4 master mixes. TCRB Tubes A and B target framework regions within the variable region, and the joining region (Vβ) of the TCR beta chain locus. TCRB Tube C targets the diversity and joining (Jβ) regions of the TCR beta chain locus. The Specimen Control Size Ladder master mix included 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-0009 Clonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-0004 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 TCRB Tube A Multiple Vβ + Jβ1/2 1 x 1500 μL tube 10 x 1500 μL tubes TCRB Tube B Multiple Vβ + Jβ2 1 x 1500 μL tube 10 x 1500 μL tubes TCRB Tube C Multiple Dβ + Jβ1/2 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 a 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 9-205-0010 IdentiClone™ TCRB Clonality Assay - Gel Detection 33 reactions 9-205-0020 IdentiClone™ TCRB Clonality Assay MegaKit - Gel Detection 330 reactions 9-205-0011 IdentiClone™ TCRB Clonality Assay - ABI Fluorescence Detection 33 reactions 9-205-0021 IdentiClone™ TCRB 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. Figure Legend: Simplified diagram of a representative rearranged T-cell receptor beta gene showing the approximate placement of the upstream and downstream DNA primers. The numbers of primers and their specificity are listed for Master Mix Tubes A, B, and C. This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.