n Medical Technology SA - FLT3 ITD detection : a closer look at the options : peer reviewed technical report




The presence of internal tandem duplication (ITD) mutations in has prognostic value in AML patients with normal cytogenetic profiles. There are several methods that can be utilized to detect these mutations with the tendency these days towards using expensive capillary analyzers and real-time amplification instruments, which prohibit ITD detection in more basic molecular facilities. We evaluated the use of a modified agarose gel electrophoresis assay for the detection of ITD mutations, in comparison to capillary electrophoresis and high resolution melt (HRM) assays, to assess the clinical diagnostic potential of this simpler technique. Each technique was evaluated on detection sensitivity, accuracy, reagent costs and processing time. Overall, in spite of the lure of advanced technology to provide faster and more sensitive analysis, in the case of ITD detection, basic molecular methodology proved to be the most sensitive, the most cost-effective and was able to deliver a reportable result in a time comparable to capillary electrophoresis. Under our test conditions, the HRM assay was neither reliable nor clinically sensitive enough for the routine detection of these mutations. This study showed that basic agarose electrophoresis methodology can successfully be employed to generate clinically relevant data regarding the ITD status of AML patients, allowing smaller molecular laboratories to offer this important prognostic assay.


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