78 Gel & Capillary | IVD Assays Invivoscribe 2019 | 79 Gel & Capillary IVD Assays FLT3 Mutation Assay - Gel Detection Assay Description The LeukoStrat® FLT3 Mutation Assay 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 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 prognosis. Assessment of the mutation status of the FLT3 (fms related tyrosine kinase 3) receptor gene in karyotype normal AML is the most important prognostic indicator of disease outcome, which is often substantial, as many studies in AML have shown that the presence of FLT3 activating mutations portends a poor prognosis1, 2 . For this reason, FLT3 activation mutation testing is required to stratify disease and determine appropriate treatment options. This LeukoStrat PCR assay targets regions of the FLT3 gene to identify ITD mutations and TKD mutations, such as the D835 and I836 mutations. This assay cannot reliably detect FLT3 mutations comprising less than 5% of the total cell population. It should be emphasized that the results of molecular mutation tests should always be interpreted in the context of clinical, histological and immunophenotypic data. This test kit includes 3 master mixes. FLT3 ITD Master mix tests for internal tandem duplication mutations. FLT3 D835 Master Mix tests for TKD region mutations. The Specimen Control Size Ladder master mix targets multiple genes and generates a series of amplicons of approximately 100, 200, 300, 400, and 600 basepairs to ensure that the quality and quantity of input DNA is adequate to yield a valid result. Performance Characteristics This LeukoStrat FLT3 Mutation Assay offers a rapid and reliable method for detecting FLT3 mutations. This is shown by a validation performed by the Laboratory for Personalized Molecular Medicine (LabPMM). Concordance with three independent labs: ITD: 100% sensitivity / 100% specificity TKD: 100% sensitivity / 100% specificity LabPMM tested 57 blinded patient samples obtained from three independent institutions. The institutions determined that 13 of the samples were FLT3 ITD positive, 33 were FLT3 ITD negative, 6 were FLT3 TKD positive, and 50 were FLT3 TKD negative. In addition 10 positive blinded spiked samples and 10 negative samples were used for the validation of FLT3 TKD. The LeukoStrat FLT3 Mutation Assay showed a sensitivity and specificity of 100% with both master mixes. The analytical sensitivity of both master mixes was determined to be 5 positive cells out of 100 total cells. 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). Reagents Controls Concentration Units in 33 Reaction Assay Units in 330 Reaction Assay IVS-0050 Clonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-P004 Clonal Control DNA 171 pg/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 33 Reaction Assay Units in 330 Assay MegaKit FLT3 ITD Master Mix - Unlabeled FLT3 ITD 1 x 1500 μL tube 10 x 1500 μL tubes FLT3 ITD Master Mix - Unlabeled FLT3 TKD 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 Agarose gel electrophoresis is used to resolve the 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 9-412-0010 LeukoStrat® FLT3 Mutation Assay - Gel Detection 33 reactions 9-412-0020 LeukoStrat® FLT3 Mutation Assay MegaKit - Gel 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