72 Gel & Capillary | CE-IVD Assays Invivoscribe 2019 | 73 Gel & Capillary CE-IVD Assays BCL1/JH Translocation Assay Assay Description The IdentiClone BCL1/JH Translocation Assay is an in vitro diagnostic product intended for PCR-based detection of BCL1/JH t(11;14)(q13;q32) gene translocations in patients with suspect lymphoproliferations. Specifically, the BCL1/JH Translocation Assay can be used to: • Identify BCL1/JH gene translocations highly suggestive of mantle cell lymphoma (MCL) •  Distinguish mantle cell lymphoma from other neoplastic or benign B-cell proliferations • 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 includes 2 master mixes. The BCL1/JH Tube targets the major translocation cluster (MTC) of the IGH-CCND1 locus and the joining region of the immunoglobulin heavy chain locus. 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 assay analytical performance was evaluated by testing spiked Mantle Cell Lymphoma (MCL) IGH-CCND1 positive cell-line DNA into tonsil DNA at six different dilutions. The Limit of Detection (LoD) was observed at 0.1% DNA dilution. To evaluate within-laboratory precision, complete agreement of results was observed across four runs executed by two operators over two days. Testing conducted across three laboratories using 25 samples from cases of MCL with IGH-CCND1 translocations and 18 negative samples, showed 100% concordance of positive samples (25 of 25 samples) using fluorescence detection, and 88% (22 of 25 samples) using gel detection. For the negative samples, the concordance was 100% using both gel detection (18 of 18 samples) and fluorescence detection (18 of 18 samples) formats. Specificity for both formats was 100% and sensitivity was determined to be between 10-3 and 10-4 . The sensitivity is sufficiently high for the detection of the IGH- CCND1 breakpoint in diagnostic material. However, only 40-50% of the t(11;14) breakpoints in MCL will be detected by PCR alone and additional detection method tools are recommended for diagnosis of breakpoints that do not fall within the major translocation cluster region. Reference 1.  JJM van Dongen et al., Leukemia 17:2257-2317 (2003). Reagents 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-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 BCL1/JH Tube - Unlabeled MTC of BCL1 + 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 Ordering Information Catalog # Products Quantity 9-308-0010 IdentiClone™ BCL1/JH Translocation Assay - Gel Detection 33 reactions 9-308-0020 IdentiClone™ BCL1/JH Translocation Assay MegaKit - Gel Detection 330 reactions 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. The use of a DNA ladder allows the relative size of amplicons to be determined. These are in vitro diagnostic products, and are not available for sale or use within North America. Figure Legend: Schematic diagram of the IGH-CCND1 t(11;14) translocation showing the cyclin D1 (CCND1) gene on the left and the Ig heavy chain (IGH) gene on the right. Shown are the relative positions and orientations for the BCL1/MTC primer and the JH primer, which are included in the BCL1/JH Master Mix tube. This assay is based on the EuroClonality/BIOMED-2 Concerted Action BMH4-CT98-3936.