One Target, Different Results: The Clinical Impact of Diagnostic Kit Choice in BCR::ABL1 Testing for Chronic Myeloid Leukemia.

Background: Quantitative PCR measurement of BCR::ABL1 is essential for monitoring molecular response and detecting relapse in chronic myeloid leukemia (CML) patients. Given the availability of multiple commercial kits for cDNA synthesis and minimal residual disease assessment, analytical accuracy and reliability are critical. This study evaluated two commercial RT and qPCR kits for BCR::ABL1 quantification and fusion transcript variant identification. Methods: Total RNA was isolated from peripheral blood, bone marrow, and external quality control samples from the UK NEQAS for Leucocyte Immunophenotyping program. cDNA synthesis was performed using two kits: AffinityScript (ASK) and RT Kit (RTK). BCR::ABL1 transcript levels were determined using the ipsogen® BCR-ABL1 Mbcr IS-MMR Kit (IPS) and the LightMix® bcr-abl t(9;22) M/m/µ Kit (TMB). Results were compared with UK NEQAS LI reference data. Fusion transcript variants were analyzed using nested PCR and a commercial qPCR assay. Results: Substantial variability was observed between the TMB and IPS assays, with moderate, non-significant correlation and wide limits of agreement. Established discrepancies resulted in different classifications of molecular response, and IPS results showed better concordance with external quality assessment data. ABL1 quantification revealed significantly higher copy numbers with the RTK compared to the ASK (p < 0.0001), enabling more reliable assessment of deep molecular responses. Statistical analyses indicated systematic and proportional bias between the methods. For variant detection, nested PCR demonstrated higher specificity, while the commercial assay showed limited discriminatory capability. Conclusions: Significant methodological differences may affect clinical interpretation, underscoring the importance of validation and standardization in CML molecular monitoring.