FLT3 Mutation Assay - ABI Fluorescence Detection

Product Details

• Summary of Explanation of the Test

  BACKGROUND:

  Acute myeloid leukemia (AML) in general has a poor prognosis. Recent studies have described mutation of the FLT3 (fms-like tyrosine kinase 3) receptor to be the most important prognostic factor in AML, with FLT3 mutants having a worse outcome and response to standard chemotherapeutic interventions. Accordingly, identification of an FLT3 mutation in AML may indicate a need to reassess and modify standard treatment options.

  The FLT3 gene (aliases: STK1; CD135; FLK-2) contains 24 exons and spans at least 96 kb. FLT3 is a receptor tyrosine kinase that is normally expressed on many cell types including hematologic stem cells.

  All types of AML can have activating mutations in the FLT3 gene. Mutation of the FLT3 receptor, either by internal tandem duplication (ITD) of the juxtamembrane domain or by point mutation of the aspartic acid residue D835 in the activation loop of the kinase domain, causes constitutive activation of the FLT3 receptor.

  Gel electrophoresis is commonly used to resolve the different-sized amplicon products and ethidium bromide or other DNA intercalating dyes to stain and detect these products. A powerful alternative method is use of differential fluorescence detection with primers conjugated with fluorescent dyes that correspond to different targeted regions. Reaction products from several different master mixes can be pooled, fractionated using capillary electrophoresis, and detected simultaneously. This detection system results in unsurpassed sensitivity, single base resolution, differential product detection and relative quantification. In addition, the laboratory can eliminate the use of agarose and polyacrylamide gels, as well as the use of carcinogens such as ethidium bromide. Further, differential detection allows accurate, reproducible and objective interpretation of primer-specific products and automatic archiving of data. The limit of detection of this assay has been determined to be approximately 1 clonal cell in 100 hundred normal cells, and inter-assay and intra-assay reproducibility in size determination using capillary electrophoresis is approximately a single basepair. This reproducibility and sensitivity allows monitoring and tracking of individual tumors during research or methods development. The automatic archiving of specimen data allows comparison of data collected at different times.

  This test kit includes 3 master mixes. The ITD and D835 master mixes target the juxtamembrane and kinase domain regions (respectively). The third master mix, the Specimen Control Size Ladder, targets multiple genes and generates a series of amplicons of 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 standard assays. Many of our customers have remarked that this improves consistency and facilitates cross training on a broad range of different assays. These robust Invivoscribe assays can be used to test DNA extracted from virtually any source.

• Specimen Requirements

  This assay tests genomic DNA

  • 5 cc of peripheral blood, bone marrow biopsy, or bone marrow aspirate anti-coagulated with heparin or EDTA. Ship at ambient temperature; OR
  • Minimum 5mm cube of tissue shipped frozen; or at room temperature or on ice in RPMI 1640; OR
  • 2 µg of genomic DNA; OR
  • Formalin-fixed paraffin embedded tissue or slides.