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CAT #: 94120101

LeukoStrat® FLT3 Mutation Assay 2.0 MegaKit – ABI Fluorescence Detection

Intended Use

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.

Product Details

  • Summary of Explanation of the Test

    Acute myeloid leukemia (AML) in general has a poor prognosis. Assessment of the mutation status of the FLT3 (fms like 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 internal tandem duplication (ITD) mutations and tyrosine kinase domain (TKD) mutations, such as the D835 and I836.

    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 2 main master mixes: the FLT3-ITD Master Mix for the detection of internal tandem duplication mutations and FLT3-D835 Master Mix for the detection of TKD region mutations (such as the D835 and I836 mutations).


    References: 

    1. Murphy, KM. et al., (2003). Detection of FLT3 Internal Tandem Duplication and D835 Mutations by a Multiplex Polymerase Chain Reaction and Capillary Electrophoresis Assay. The Journal of Molecular Diagnostics 5, 96 – 102.

    2. Yamamoto, Y. et al., (2001). Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood 97, 2434-2439.

  • Principles of the Procedure

    Internal Tandem Duplication (ITD) Mutations of FLT3

    FLT3 internal tandem duplication or length mutations are caused by duplication and insertion of a portion of the FLT3 gene that includes the region in and around the juxtamembrane (JM) region of the FLT3 gene. These mutations vary in both the location and the length of the inserted duplicated DNA sequence. ITD mutations result in constitutive autophosphorylation and activation of FLT3. Wild-type FLT3 alleles will amplify and produce a 327 bp product using this assay, while alleles that contain ITD mutations will produce a product ≥330 bp.

     

     

     

     

     

     

     

     

     

     

    Depicted is a representation of the FLT3 JM region and the activating loop of the kinase domain. Green and blue dots with black arrows represent the relative positions of primers that target the JM region for ITD and yellow dots with black arrows represent the relative positions of the primers that target TKD mutations in the activating loop of the kinase domain. The yellow box has vertical black lines that represent the position of the wild-type EcoRV restriction digest sites.

    Tyrosine Kinase Domain (TKD) Mutations of FLT3

    FLT3 tyrosine kinase domain (TKD) mutations are caused by nucleic acid substitutions that result in a change in the amino acid sequence in this highly conserved catalytic center. TKD mutations, such as D835 and I836, result in constitutive autophosphorylation and activation of FLT32. Wild-type alleles of the FLT3 gene include an EcoRV restriction digest site. When a nucleic acid substitution occurs, the restriction digest recognition site disappears, and the EcoRV endonuclease is unable to identify and digest the DNA at this site. Our tests use primers that lie on either side of the TKD region. The FLT3 target region is amplified using PCR and then an EcoRV restriction digest is performed. One of the PCR primers contains an EcoRV restriction site, so both wild type and mutant alleles are digested. The digestion pattern identifies loss of the normal gene sequence and ensures that digestion occurred. Wild-type alleles of the FLT3 gene yield products of 79 bp and mutant alleles yield products of 127 bp. Undigested amplicons are 147 bp (product lengths correspond to results obtained through the use of GeneScan™ – 600 LIZ® Size Standard v2.0 and the ABI3500xL instrument. The use of different size standards and instruments may yield different product sizes).

    Differential Fluorescence Detection

    Differential fluorescence detection is commonly used to resolve the different-sized amplicon products using a capillary electrophoresis instrument. Primers can be conjugated with several different fluorescent dyes (fluorophors) so that they can 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 unsurpassed sensitivity, single nucleotide resolution, differential product detection, and relative quantification. In addition, the use of agarose and polyacrylamide gels, as well as the use of carcinogens such as ethidium bromide, can virtually be eliminated. Further, differential detection allows accurate, reproducible and objective interpretation of primer-specific products and automatic archiving of data. Inter-assay and intra-assay reproducibility in size determination using capillary electrophoresis is approximately 1 to 2 nucleotides. This reproducibility and sensitivity coupled with the automatic archiving of specimen data allows for the monitoring, tracking, and comparison of data from individual patients over time.

  • Specimen Requirements

    This assay tests genomic DNA from the following sources:

    • Peripheral blood or bone marrow aspirate anti-coagulated with heparin, EDTA, ACD or previously isolated mononuclear cells that are fresh in an appropriate media (RPMI or similar) or frozen in an appropriate cryopreservation media.
    • Peripheral blood and bone marrow aspirates may be stored at 2°C to 8°C for 7 days and still give valid results. Isolated mononuclear cells may be stored fresh for up to 7 days or indefinitely if properly cryropreserved.
    • 500 ng of genomic DNA (stored at 2°C to 8°C or below -15°C and shipped at ambient temperature, cool conditions, or on dry ice).

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