Next-generation digital polymerase chain reaction assay for BCR::ABL1 in chronic myeloid leukemia: comprehensive validation for standardized measurable residual disease detection.

purposeThe sensitivity of real-time quantitative reverse-transcription polymerase chain reaction (RT-qPCR) for detecting measurable residual disease (MRD) in patients with chronic myeloid leukemia (CML) who have achieved a deep molecular response is limited. We validated a digital PCR (dPCR) assay (LQ Check BCR::ABL1 Major %IS Kit) for MRD quantification.

methodsValidation was performed according to CLSI guidelines EP17-A2, EP06, EP05-A3, and EP07 using BCR::ABL1-positive and -negative materials and the World Health Organization (WHO) 1st International Standard. A nonparametric analysis of the limit of blank was used to establish a positivity cutoff of ≥ 2 droplets. The limit of detection (LoD) was verified using a detection criterion of ≥ 95%, and the limit of quantitation (LoQ) was defined as the lowest level meeting ≥ 95% detection and a study-defined %CV of ≤ 76%. Clinical validation included 72 CML specimens for molecular response (MR) category comparisons and 67 specimens for paired quantitative comparisons.

resultsThe LoD and LoQ were both 0.0025% on the International Scale (IS) with a detection rate of 95.8%. The assay showed linearity over 5 logs (R

conclusionThe LQ Check BCR::ABL1 Major %IS Kit enabled sensitive, precise, and reproducible BCR::ABL1 quantification at MR4.6. The clinical significance of a deeper MR classification using dPCR, particularly for treatment-free remission, requires longitudinal validation.