Establishment of genomic RNA reference materials for BCR‑ABL1 P210 measurement

Yi Yang ^{1 2}, Xia Wang ¹, Chunyan Niu ¹, Shujun Zhou ², Huafang Gao ^{3 4}, Xiaohua Jin ^{3 4}, Shangjun Wang ⁵, Meihong Du ⁶, Xiaoyan Cheng ⁶, Lingxiang Zhu ⁷, Lianhua Dong ⁸

Abstract

Quantitation of BCR-ABL1 by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) is vital for monitoring chronic myeloid leukemia (CML), and relevant detection depends on RNA reference materials. A genomic RNA reference material (RM) carrying the BCR-ABL1 P210 fusion mutation was prepared. A one-step reverse transcription digital PCR (RT-dPCR) based absolute quantification method was established to characterize the RM. The dPCR method presents high accuracy and favorable analytical sensitivity. Satisfactory linear correlation was observed between measured and nominal values of b2a2, b3a2 and ABL1-ref within the dynamic range of 10¹–10⁴ copies/reaction, with 0.94 < slope < 1.04 and R²≥0.99. Homogeneity and stability tests confirmed uniform properties of the RM, which remains stable for 24 months at -80 °C. Inter-laboratory reproducibility was evaluated across eight laboratories. Consistent quantification results of b3a2, ABL1-ref and BCR-ABL1/ABL1 ratio were obtained, with CV < 2.0 %. The RM can facilitate metrological traceability establishment for BCR-ABL1 fusion gene mutation detection and support routine quality control in testing laboratories.