Qualitative PCR Detection of FLT3-ITD and TKD Mutations in Acute Myelogenous Leukemia

The TRUPCR® FLT3 Mutation Detection Kit is a sensitive, CE-IVD certified in vitro diagnostic assay developed for the qualitative detection of FLT3 internal tandem duplications (ITD) and tyrosine kinase domain (TKD) point mutations (D835-I836) in genomic DNA isolated from mononuclear cells obtained from peripheral blood. The kit combines both conventional PCR (for ITD) and real-time PCR (for TKD mutations), offering comprehensive mutation coverage in a single test run. It is designed to aid in the diagnosis, risk stratification, and therapeutic decision-making in patients with acute myelogenous leukemia (AML).

*Please Note: This kit is also available with the TKD mutation only*

Molecular Background

Acute myelogenous leukemia (AML) is a clonal hematologic malignancy marked by the accumulation of immature myeloid cells in the bone marrow and blood. Among the most frequently mutated genes in AML is FLT3, which encodes a receptor tyrosine kinase (RTK) involved in cell proliferation, differentiation, and survival through critical intracellular signaling pathways.

FLT3 mutations are found in approximately 30% of AML cases, with two major mutation types of clinical importance:

• Internal Tandem Duplication (ITD): Occurs in the juxtamembrane domain between exons 14 and 15. These mutations vary in size and are associated with poor prognosis, high relapse rates, and shortened survival.

• Tyrosine Kinase Domain (TKD) mutations: Most often at codon D835, typically a point mutation from GAT to TAT (D835Y), leading to constitutive activation of the receptor due to loss of autoinhibition.

Detection of FLT3 mutations is essential in AML diagnosis and classification, especially given the availability of FLT3-targeted therapies and their use in risk-adapted treatment strategies.

Assay Performance & Utility

The TRUPCR® FLT3 Mutation Detection Kit enables accurate, sensitive detection of both ITD and TKD mutations in a single workflow. With a detection limit of 1% mutant allele and the ability to detect ITDs as short as 20 base pairs, the assay supports:

• Early identification of high-risk AML subtypes
• Comprehensive mutation profiling, covering both major FLT3 mutation types
• Stratification of patients for targeted therapy and treatment response monitoring

The kit is optimized for routine diagnostic use in clinical molecular laboratories, offering high accuracy, ease of use, and consistent performance across compatible instruments.

Technical Specifications

The TRUPCR® FLT3 kit employs a dual PCR strategy—conventional PCR for detection of FLT3-ITDs and TaqMan-based real-time PCR for D835/TKD mutations. It is designed to work with genomic DNA extracted from mononuclear cells, with an input range of 50–200 ng per reaction.

The assay delivers results in approximately 90 minutes (real-time) and ~2 hours for conventional PCR. It is validated for use with a wide range of qPCR platforms, including ABI 7500, Bio-Rad CFX96™, Roche LightCycler®, and standard thermal cyclers.

All reagents are supplied in a ready-to-use format, minimizing setup time and reducing the potential for user error. Built-in controls—Positive Control, Negative Control, and No Template Control (NTC)—ensure result reliability and validation of each run.

The kit is stored at –20°C and has a shelf life of 12 months from the manufacturing date. It is CE-IVD certified and intended for use by certified diagnostic laboratories.