Comparison of digital PCR with real-time PCR calibrated to the International Scale for BCR-ABL monitoring.

Abstract

e18545 Background: Monitoring BCR-ABL1 fusion transcripts is the cornerstone of management in chronic myeloid leukemia (CML). Real Time PCR(RTPCR) has been the tool of choice for its sensitivity and dynamic range. The International Scale was recently introduced to allow for standardization across laboratories. Chip based Digital PCR(dPCR) which allows for absolute quantitation may obliviate the need for such standardization by giving absolute results.It has the potential to increase sensitivity for detection of minimal residual disease. This assumes great significance as we move into the era of treatment free remission. Methods: A total of 31 EDTA-blood samples of CML patients with known values ranging from 0.003-0.5 IS were processed via an accredited RTPCR protocol calibrated to the IS scale and a parallel dPCR workflow without any specific calibration . The analysis of digital PCR samples was performed on the Thermofisher Cloud Platform. Results: Both RTPCR and dPCR yielded extremely concordant results. The Pearson correlation between the two methods was r = 0.6043 (95%CI: 0.318 to 0.789; p = 0.0003). The calculated BCR-ABL/ABL ratios were comparable with a median of 0.098 for RT-PCR calibrated to the IS (range 0.003-0.55; n = 26) and 0.11 for dPCR (range 0.003-0.37; n = 29). The mean difference for the ratios between the two methods used for the detection was around 0.08. Conclusions: We demonstrate here the capability of dPCR to provide a parallel result to an IS scale calibrated protocol without any standardization. This approach required no specific calibrators or standards and resulted in extremely cost effective testing. This freedom from routine calibration provides for a significantly more robust workflow and greatly increased reliability. Limitations do persist in dPCR on account of limited chip densities which are the main parameter for the Poisson statistics. This has limited the dynamic range on dPCR to a maximum of Log 4 for accurate quantification. However as chip densities and emulsion concentrations increase in these technologies, they are poised to introduce a new era in the quest of accurate quantification.

Overview of Molecular Quantification of the BCR-ABL Oncogene in CML Patients

“…qPCR is considered as an effective technique and actual sensitive method for distinguishing and measuring the quantity of BCR-ABL gene 30 . Using bone marrow or blood sample, More further, qPCR technique is able to detect a low amounts of the BCR-ABL gene particularly when Philadelphia chromosome cannot be identified in blood or bone marrow cells in cytogenetic testing 31 . The BCR-ABLE qPCR assay is a fundamental tool for aiding in CML diagnosis from the genetic side 32 .…”