MRD was followed up with NPM1 or WT1 RT-qPCR; NPM1 expression was analyzed after allo-HSCT by RT-qPCR in those patients with NPM1-mutated AML. In the patients without NPM1 mutations, WT1 expression was measured through RT-qPCR after allo-HSCT. All RT-qPCR tests were performed in a LightCycler 1.5 (Roche, Switzerland) using specific probes and primers, as previously described [15 (link),16 (link)]. MRD was also analyzed by multiparametric flow cytometry (MFC) in every BM sample during follow-up using monoclonal antibodies corresponding to the immunophenotypic profile identified in the diagnosis of each patient, using a DxFLEX cytometer (Beckman Coulter, Brea, CA, USA).
Chimerism analysis by STR-PCR was performed using the AmpFlSTR™ SGM Plus™ PCR Amplification Kit (Applied Biosystems) and the Mentype®Chimera® kit (Biotype, Dresden, Germany), which included polymorphic, autosomal, non-coding STR loci (10 for SGM Plus™ and 12 for Chimera®) and Amelogenin as a sex-specific marker. PCR was carried out using specific primers fluorescence-labeled with 6-FAMTM, BTG or BTY in a GeneAmp® PCR System 9700 Thermal Cycler (Applied Biosystems, USA), followed by capillary electrophoresis using an ABI3130xl DNA sequencer (Applied Biosystems). Electropherograms were analyzed through GeneMapper™ Software 5 (Thermo Fisher, USA).
Chimerism analysis by STR-PCR was performed using the AmpFlSTR™ SGM Plus™ PCR Amplification Kit (Applied Biosystems) and the Mentype®Chimera® kit (Biotype, Dresden, Germany), which included polymorphic, autosomal, non-coding STR loci (10 for SGM Plus™ and 12 for Chimera®) and Amelogenin as a sex-specific marker. PCR was carried out using specific primers fluorescence-labeled with 6-FAMTM, BTG or BTY in a GeneAmp® PCR System 9700 Thermal Cycler (Applied Biosystems, USA), followed by capillary electrophoresis using an ABI3130xl DNA sequencer (Applied Biosystems). Electropherograms were analyzed through GeneMapper™ Software 5 (Thermo Fisher, USA).