The interrogation of FLT3 detects internal tandem duplications (ITDs) and tyrosine kinase domain (TKD) mutations in the FLT3 gene using polymerase chain reaction (PCR) coupled with fragment analysis by capillary electrophoresis.11,12 The analytical sensitivity is 5% mutant allele fraction within the total cell population.
The interrogation of IDH1/IDH2 detects potential mutations in the entire coding regions of IDH1 exon 4 and IDH2 exon 4. These exons are amplified, and the PCR products are purified and sequenced in both forward and reverse directions by dye-terminator Sanger sequencing on an automated platform. The analytical sensitivity is 20% mutant allele fraction within the total cell population.
The remaining genes are interrogated by NGS. Clinically relevant regions in CEBPA (entire coding region), KIT (exons 2, 3, 8-11, 13, 14, 17-19), NPM1 (exon 11), and TP53 (all coding exons), are interrogated for potential mutations by a bait-capture–based massively parallel sequencing test methodology using the Illumina NextSeq platform. The analytical sensitivity of the assay is 5% mutant allele fraction within the total cell population for both single nucleotide variants and small indels. In accordance with the Association for Molecular Pathology (AMP) and American College of Molecular Genetics (ACMG) guidelines, benign or likely benign variants will not be reported from this assay.