Myeloid neoplasms comprise a heterogeneous group of hematologic malignancies characterized by the accumulation of genetic alterations that influence diagnosis, prognosis, and therapeutic decision-making. Among these alterations, FLT3 internal tandem duplications (FLT3-ITD) are of major clinical relevance, particularly in acute myeloid leukemia, where their detection is an essential component of the molecular characterization of the disease. However, more comprehensive approaches, such as next-generation sequencing (NGS), remain limited by their cost and availability in many diagnostic laboratories, highlighting the need for accessible, sensitive, and reproducible methodologies.

The present study aimed to standardize a polymerase chain reaction (PCR)-based fragment analysis assay using capillary electrophoresis for the detection of FLT3-ITD mutations and to apply it to 96 patients with different myeloid neoplasms from the northwestern region of Paraná, Brazil. Initially, two primer pairs previously described in the literature were compared during the standardization process, and only the primer pair showing the best analytical performance was selected for subsequent analyses. The analytical sensitivity of the assay was also evaluated using serial dilutions of the PCR product obtained from an FLT3-ITD-positive control sample.

Fragment analysis identified FLT3-ITD mutations in 7 of the 96 samples analyzed, corresponding to a frequency of 7.3% in the study population. Different duplication lengths were detected, and in some cases, multiple peaks consistent with clonal heterogeneity were observed. The serial dilution assay demonstrated persistent detection of the mutant fragment in all evaluated dilutions, up to 1:200, with fluorescence intensity remaining above the recommended analytical threshold, confirming the adequate analytical sensitivity of the method. These findings demonstrate that PCR-based fragment analysis using capillary electrophoresis has the potential to expand access to molecular diagnosis, particularly in healthcare settings with limited access to more complex molecular techniques, as is frequently the case in the Brazilian Unified Health System (SUS).