Madalina CIRNU, Dan-Cristian MARINESCU, Cosmin-Florentin NICULAE, Stefania-Andreea NEGRU, Andreea-Alexandra URECHE-FOTEA, Dan-Sebastian SOARE, Horia BUMBEA, Eugen RADU
Background: Accurate quantification of BCR::ABL1 transcripts is paramount for the management of chronic myeloid leukemia (CML) and Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL). Despite advancements in real-time reverse transcription PCR (RT-qPCR), the need for more sensitive and precise assays remains. This study evaluated the diagnostic utility of digital reverse transcription PCR (dRT-PCR) for BCR::ABL1 quantification in assessing minimal residual disease (MRD).
Material and methods. Twelve patients with CML or Ph+ ALL were included. Peripheral blood or bone marrow samples, collected in PaxGene RNA preservation tubes, were extracted with the automated QIASymphony SP instrument. dRT-PCR conducted on the LOAA analyzer, utilized the Dr. PCR BCR-ABL1 Detection Kit CE_IVD (OPTOLANE Technologies, South Korea). By employing a dual-labeled hydrolysis probe and target-specific primers, this protocol enabled the simultaneous amplification and detection of BCR::ABL1 transcripts e13a2 and e14a2 and ABL1. The resulting copy numbers/reaction were used to calculate the International Scale percentage (%IS) and the molecular reduction (MR) value and analyzed for clinical correlations.
Results. Digital RT-PCR demonstrated high precision in quantifying BCR::ABL1 transcripts, the lowest %IS value being 0.02%. A consistent quality RNA sample totalizing 110 ng/reaction was crucial for successful reactions. This assay proved faster and easier than conventional RT-PCR and did not require standards or calibrators.
Conclusion. This study supports the feasibility of digital RT-PCR for quantitative analysis. Future objectives for our laboratory include expanding our diagnostic repertoire using this platform, for enhancing patient care
https://doi.org/10.59854/dhrrh.2024.2.2.81
