ENHANCED DETECTION AND MONITORING OF CML USING MULTIPLEX REAL-TIME PCR WITH MAGNETIC RNA EXTRACTION

Abstract

Introduction: Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of a BCR-ABL1 rearrangement. Its detection and quantification by real-time quantitative PCR (RQ-PCR) play a central role in CML diagnosis, therapy monitoring, and sequencing to identify resistance mutations in case of therapy failure. As previously demonstrated (Pugliesi et al., Hematol Transfus Cell Ther. 2023;45 Suppl 4), combining magnetic RNA extraction with the use of only 2mL of peripheral blood (PB) provides a fast, cost-effective, and highly sensitive strategy for CML diagnosis and monitoring. Objectives: To optimize the existing method by integrating magnetic RNA extraction with multiplex real-time PCR amplification for both the BCR (internal control) and BCR::ABL1 p210 transcript within the same PCR reaction. This enhancement aims to increase scalability and traceability while reducing costs and turnaround time (TAT). Materials and methods: Total RNA was extracted from 100 PB samples. After erythrocyte lysis, leukocytes from 2mL of PB were automatically extracted using Extracta-MPTA (Loccus do Brasil). To validate a multiplex assay combining the BCR gene and the target BCR::ABL1, the reference standard curve ERM-AD263 was used to calibrate the assay, and a commercial reference RNA was used to set the International Scale (IS%). BCR and BCR::ABL1 were measured using previously validated singleplex assays and by multiplex (BCR and BCR::ABL1 in the same reaction). Multiplex assay accuracy was measured by comparing it to the previously validated singleplex real-time PCR for BCR and BCR::ABL1 copy number and BCR::ABL1 ratio (IS%) using 100 CML samples (paired t-test). Precision was determined using internal quality control (IQC) (BCR::ABL1: 1%) in duplicates in 20 independent assays. Results: The singleplex and multiplex five-point standard curves for BCR and BCR::ABL1 were completely superposed (Spearman correlation, p = 0.003). Comparisons in 100 samples revealed that BCR and BCR::ABL1 measurements were similar (paired t-test, p = 0.42 and 0.07, respectively) and that BCR::ABL1 IS% values were identical (p = 0.18). Pairing was significantly effective for all determinations (p < 0.0001). The total coefficient of variation for internal quality control (IQC) was 25%, with a standard deviation of 0.36. Utilizing the multiplex test reduced the turnaround time (TAT) by 20%, allowing results to be released within 24 hours of sample reception. Discussion: As demonstrated, the combination of automated magnetic RNA extraction from 2mL of PB with the quantification of the BCR::ABL1 gene for patients under CML monitoring is an effective, fast, safe, and low-cost method. Conclusion: The integration of automated magnetic RNA extraction from 2mL of peripheral blood with multiplex real-time PCR for BCR::ABL1 quantification provides a robust, efficient, and economical approach for monitoring CML patients. This optimized method significantly reduces turnaround time, enabling results to be available within 24 hours of sample reception. Such a strategy is particularly advantageous for public healthcare systems, such as the Brazilian Unified Health System (SUS), enhancing accessibility and ensuring timely and accurate monitoring of CML patients. The streamlined process supports scalable and traceable diagnostics, ultimately contributing to improved patient management and outcomes.