80 Gel & Capillary | IVD Assays Invivoscribe 2019 | 81 Gel & Capillary IVD Assays FLT3 Mutation Assay 2.0 - ABI Fluorescence Detection Assay Description The LeukoStrat® FLT3 Mutation Assay 2.0 is an in vitro diagnostic product intended for PCR-based detection of FLT3 activating mutations in patients with acute myelogenous leukemia (AML). Specifically, the FLT3 Mutation Assay 2.0 can be used to: • Identify internal tandem duplications (ITD) in the FLT3 gene • Identify tyrosine kinase domain (TKD) mutations in the FLT3 gene Summary and Explanation of the Test AML in general has a poor prognosis1,2 . Assessment of the mutation status of the FLT3 (fms related tyrosine kinase 3) receptor gene is the most important prognostic indicator of disease outcome, occurring in approximately 30% of patients at the time of diagnosis3 . For this reason, testing for FLT3 activating mutations is required for the stratification of disease and determination of appropriate treatment options. The LeukoStrat FLT3 Mutation Assay 2.0 is a PCR-based method that identifies ITD and TKD mutations. This test kit includes 2 master mixes: the FLT3-ITD Master Mix for the detection of ITD mutations and FLT3-D835 Master Mix for the detection of TKD region mutations (such as the D835 and I836 mutations). Performance Characteristics The LeukoStrat FLT3 Mutation Assay 2.0 is a rapid and reliable method for the detection of FLT3 mutations, as evidenced by comparison with Roche® 454 sequencing. The FLT3 Mutation Assay 2.0 is capable of detecting FLT3-ITD and TKD mutations with excellent concordance (Table 1 and 2) and has high reliability when multiple standard laboratory variables are considered, including multiple operators, reagent lots, different ABI 3500xL instruments, and nonconsecutive testing days. * 95% of results would be expected to agree with sequencing at a rate greater than or equal to the lower limit (LL). Percent Agreement Discordance # Concordance # *95% LL Negative PA 100% 0 119 96.9% Positive PA 98.0% 4 200 95.1% Table1. FLT3 ITD Percent Agreement with 454 Sequencing * 95% of results would be expected to agree with sequencing at a rate greater than or equal to the lower limit (LL). Percent Agreement Discordance # Concordance # *95% LL Negative PA 100% 0 137 96.9% Positive PA 100% 0 240 98.5% Table2. FLT3 TKD Percent Agreement with 454 Sequencing Reference 1. Murphy KM et al., A Clinical PCR/Capillary Electrophoresis Assay for the Detection of Internal Tandem Duplication and Point Mutation of the FLT3 Gene. J. Mol. Diag. 5:96-102 (2003). 2. Yamamoto, Y., et al., Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood, 97(8):2434-9 (2001). 3. Acute Myeloid Leukemia, Clinical Practice Guidelines in Oncology, National Comprehensive Cancer Network (v.2.2014) Reagents Controls Concentration Units in Assay Units in MegaKit FLT3 ITD Positive Control 50 μg/mL 1 x 100 μL tube 5 x 100 μL tubes FLT3 D835 Positive Control 50 μg/mL 1 x 100 μL tube 5 x 100 μL tubes FLT3 Negative Control 50 μg/mL 1 x 100 μL tube 5 x 100 μL tubes Master Mixes Target Units in Assay Units in MegaKit FLT3 ITD Master Mix – 6FAM & HEX FLT3 ITD 1 x 1500 μL tube 10 x 1500 μL tubes FLT3 D835 Master Mix – 6FAM FLT3 TKD 1 x 1500 μL tube 10 x 1500 μL tubes 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 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 high 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. The data shown was generated using the master mixes indicated. Amplified products were run on an ABI 3500xL instrument. Ordering Information Catalog # Products Quantity 9-412-0091 LeukoStrat® FLT3 Mutation Assay 2.0 – ABI Fluorescence Detection 33 reactions 9-412-0101 LeukoStrat® FLT3 Mutation Assay 2.0 MegaKit – ABI Fluorescence Detection 330 reactions These are in vitro diagnostic products, and are not available for sale or use within North America. Gel & Capillary CE-IVD Assays