Droplet digital PCR for quantification of PML-RARα in acute promyelocytic leukemia: a comprehensive comparison with real-time PCR
Real-time quantitative PCR (qPCR) has been widely implemented for molecular testing, but there are still some inherent limitations that hamper its usefulness. Droplet digital PCR (ddPCR), which can provide direct, standards-free quantification, has recently received increasing attention. In our study, a comprehensive comparison of ddPCR with qPCR in relation to the quantification of PML-RARα was performed to evaluate the diagnostic potential of ddPCR. Results showed that ddPCR displayed significant concordance with qPCR in the detection of PML-RARα in clinical samples, but showed advantages over qPCR in terms of precision, limit of detection (LOD), and other basic performance parameters. A study of the feasibility of duplexing also indicated that ddPCR could simultaneously quantify the target PML-RARα and the clinical common reference gene ABL in a reaction, in contrast to qPCR. Moreover, ddPCR was more tolerant than qPCR of inhibition, and was shown to be able to quantify inhibition-prone samples. Another advantage of using ddPCR in clinical applications is that it will yield accurate results for patients with PML-RARα levels that fluctuate around the LOD of qPCR. Therefore, ddPCR is considered to have the potential to become a reliable alternative technique for quantifying PML-RARα.
KeywordsddPCR qPCR PML-RARα quantification Comparison of methods
This study was supported by the National Natural Science Foundation (grant numbers 81201349, 81000775); Young Medical Key Talents in Jiangsu Province (grant numbers QNRC 2016686, 2016687); Frontier and Key Technical Innovation Projects of Nantong (grant number MS22015049); and the Nantong Science and Technology Plan Project (MS12017008-3).
Compliance with ethical standards
The authors declare that all individual participants from whom the blood samples were obtained gave their informed consent, and that the studies were approved by the ethics committee of the Affiliated Hospital of Nantong University and were performed in accordance with ethical standards.
Conflicts of interest
The authors declare no conflict of interest.
- 2.Lo-Coco F, Ammatuna E. The biology of acute promyelocytic leukemia and its impact on diagnosis and treatment. Hematol Am Soc Hematol Educ Program. 2006;156-61:514.Google Scholar
- 3.Lo Coco F, Diverio D, Falini B, Biondi A, Nervi C, Pelicci PG. Genetic diagnosis and molecular monitoring in the management of acute promyelocytic leukemia. Blood. 1999;94(1):12–22.Google Scholar
- 12.Brunetti C, Anelli L, Zagaria A, Minervini A, Minervini CF, et al. Droplet digital PCR is a reliable tool for monitoring minimal residual disease in acute promyelocytic leukemia. J Mol Diagn. 2017;19(3):437–44.Google Scholar
- 13.He HJ, Almeida JL, Lund SP, Steffen CR, Choquette S, Cole KD. Development of NIST Standard Reference Material 2373: genomic DNA standards for HER2 measurements. Biomol Detect Quantif. 2016;8:1–8.Google Scholar
- 15.Clinical and Laboratory Standards Institute, Tholen DW, Linnet K, Kondratovich M, Armbruster DA, Garrett PE, Jones RL, Kroll MH, Lequin RM, Pankratz TJ, Scassellati GA, Schimmel H, Tsai J. Protocols for determination of limits of detection and limits of quantitation. Approved guideline EP17-a. Wayne: Clinical and Laboratory Standards Institute; 2004.Google Scholar
- 16.Milosevic D, Mills JR, Campion MB, Vidal Folch N, Voss JS, et al. Applying standard clinical chemistry assay validation to droplet digital PCR quantitative liquid biopsy testing. Clin Chem. 2018.Google Scholar
- 17.Antonelli G, Padoan A, Aita A, Sciacovelli L, Plebani M. Verification of examination procedures in clinical laboratory for imprecision, trueness and diagnostic accuracy according to ISO 15189:2012: a pragmatic approach. Clin Chem Lab Med. 2017;55(10):1501–8.Google Scholar
- 20.Cao L, Cui X, Hu J, Li Z, Choi JR, Yang Q, et al. Xu F. Advances in digital polymerase chain reaction (dPCR) and its emerging biomedical applications. Biosens Bioelectron. 2017;90:459–74.Google Scholar
- 25.Cao Y, Griffith JF, Dorevitch S, Weisberg SB. Effectiveness of qPCR permutations, internal controls and dilution as means for minimizing the impact of inhibition while measuring Enterococcus in environmental waters. J Appl Microbiol. 2012;113(1):66–75.Google Scholar
- 29.Verhaegen B, De Reu K, De Zutter L, Verstraete K, Heyndrickx M, Van Coillie E. Comparison of droplet digital PCR and qPCR for the quantification of Shiga toxin-producing Escherichia coli in bovine feces. Toxins (Basel) 2016;8(5).Google Scholar
- 35.Basu AS. Digital assays part I: partitioning statistics and digital PCR. SLAS Technol. 2017;22(4):369–86.Google Scholar
- 37.Sinha M, Mack H, Coleman TP, Fraley SI. A high-resolution digital DNA melting platform for robust sequence profiling and enhanced genotype discrimination. SLAS Technol. 2018;23(6):580–91.Google Scholar