Development and validation of an in-house one-step RT-qPCR method for the quantification of the BCR::ABL1 p210...

Chronic myeloid leukemia (CML) is driven by the BCR::ABL1 fusion oncogene, with the p210 transcript accounting for over 98% of cases. RT-qPCR monitoring of the BCR::ABL1 fusion gene is the gold standard for detecting measurable residual disease (MRD) and guiding treatment-free remission (TFR) decisions. Most commercial assays are costly and limit accessibility in resource-constrained settings. We aimed to develop and validate an in-house one-step RT-qPCR assay for BCR::ABL1 p210 with high analytical sensitivity and precision. The assay was optimized and validated following CLSI guidelines. In-house plasmid standards were calibrated against internationally certified ERM-AD623 reference material. Analytical performance (specificity, sensitivity, precision, and linearity) and clinical validation were performed using 34 patient samples against the CE-IVD certified geneMAP BCR::ABL1 p210 IS-MMR kit. The assay demonstrated a low limit of detection (LoD) of 0.0030% BCR::ABL1/ABL1 ratio, corresponding approximately to an MR4.51-equivalent level, enabling reliable detection of low-level transcripts. No cross-reactivity with p190 variants was observed. Strong linearity was demonstrated across the MR1-equivalent to MR4.5-equivalent range. Precision met predefined acceptance criteria, with standard deviations below 0.28 MR units. Clinical validation demonstrated excellent correlation with the reference kit (Pearson's r = 0.979). This validated in-house assay demonstrates commercial-grade analytical performance, offering a cost-effective and accessible approach for reliable deep molecular response (DMR) assessment in local and research-oriented CML monitoring, particularly in resource-limited settings.