74 Gel & Capillary | CE-IVD Assays Invivoscribe 2019 | 75 Gel & Capillary CE-IVD Assays BCL2/JH Translocation Assay Assay Description The IdentiClone BCL2/JH Translocation Assay is an in vitro diagnostic product intended for PCR-based detection of IGH-BCL2 t(14;18) gene translocations in patients with suspect lymphoproliferations. Specifically, the BCL2/JH Translocation Assay can be used to: • Distinguish lymphoma from benign lymphoid hyperplasia •  Distinguish follicular lymphoma (FL) from other B-cell lymphomas that may have a similar appearance • 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 test kits include 4 master mixes. The BCL2/JH Translocation master mixes (BCL2/JH Tubes A, B, and C) target the joining (J) region of the immunoglobulin heavy chain (IGH) gene and distinct regions of the BCL2 gene. These master mixes are used to detect major breakpoint region (MBR) and minor cluster region (mcr) of the IGH-BCL2 t(14;18)(q32;q21) translocations. The 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 many of our assays. This improves consistency and facilitates cross-training. Performance Characteristics The initial evaluation of this assay was performed in three laboratories on DNA derived from 124 cases of follicular cell lymphoma (FCL) known to carry the t(14;18) translocation. 109 cases were identified with the IGH-BCL2 fusion gene (88%) using this PCR assay. The final testing and evaluation was done on samples in 11 independent laboratories1 . False-positive results (0.4%) were only seen in 12 of 3036 analyses. This IdentiClone BCL2/JH Translocation Assay was found to be more sensitive than Southern blot analysis. Sensitivity differed slightly between the master mixes. However, overall sensitivity for the assay was determined to be between 1 positive cell in 102 normal cells and 1 positive cell in 103 normal cells. In conclusion, we have designed and evaluated the performance characteristics of a robust three tube multiplex PCR assay in order to maximize the detection of the t(14;18) breakpoint. This strategy is capable of amplifying across the breakpoint region in the majority of cases of follicular lymphoma with a cytogenetically defined translocation. Reference 1.  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 100 μL @ 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-P002 Clonal Control DNA 100 μL @ 1600 pg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-0031 Clonal Control DNA 100 μL @ 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-0000 Polyclonal Control DNA 100 μL @ 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes Master Mixes Target Units in Assay Units in Assay MegaKit BCL2/JH Tube A - Unlabeled BCL2 MBR + IGH JH 1 x 1500 μL tube 10 x 1500 μL tubes BCL2/JH Tube B - Unlabeled BCL2 3’ MBR + IGH JH 1 x 1500 μL tube 10 x 1500 μL tubes BCL2/JH Tube C - Unlabeled BCL2 mcr + IGH JH 1 x 1500 μL tube 10 x 1500 μL tubes Specimen Control Size Ladder - Unlabeled Multiple Genes 1 x 1500 μL tube 10 x 1500 μL tubes Gel Electrophoresis Detection Gel electrophoresis, such as agarose gel electrophoresis, is commonly used to resolve different amplicon products based on their size, charge, and conformation. Since DNA is negatively charged, when an electrical potential (voltage) is applied across the gel containing PCR products, the electrical field causes the amplicons to migrate through the gel. Smaller DNA fragments are able to easily migrate through the gel matrix, whereas larger DNA fragments migrate more slowly. This causes a separation of the amplicon products based on size. Ethidium bromide or other DNA intercalating dyes can then be used to stain and detect these products in the gel. Ordering Information Catalog # Products Quantity 9-309-0020 IdentiClone™ BCL2/JH Translocation Assay - Gel Detection 33 reactions 9-309-0040 IdentiClone™ BCL2/JH Translocation Assay MegaKit - Gel Detection 330 reactions These are in vitro diagnostic products, and are not available for sale or use within North America. Figure Legend: Schematic diagram of the IGH-BCL2 t(14;18) translocation showing the BCL2 gene on the left and the Ig heavy chain (IGH) gene on the right. Shown are the relative positions and orientations for the major breakpoint region (MBR) primers, the minor cluster region (mcr) primers, and the JH primer, which are included in the 3 BCL2/JH master mix tubes. This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.