Digital PCR can provide improved BCR-ABL1 detection in chronic myeloid leukemia patients in deep molecular response and sensitivity of standard quantitative methods using EAC assays

• Published in: Practical laboratory medicine Vol. 25; p. e00210
• Main Authors: Smitalova, Dagmar, Dvorakova, Dana, Racil, Zdenek, Romzova, Marianna
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.05.2021; Elsevier
• Subjects: BCR-ABL1 monitoring; Chronic myeloid leukemia; Digital PCR; GeneXpert BCR-ABL Monitor assay; RT-qPCR; Short Communication; LOD; CML; MR; EAC; BCR-ABL1 monitoring; FPR; Digital PCR; IS; Chronic myeloid leukemia; TKI; pDNA; dPCR; qPCR; cDNA; NTC; GeneXpert BCR-ABL Monitor assay; RT-qPCR; LOB; CML, chronic myeloid leukemia; NTC, no template control; cDNA, complementary DNA; MR, molecular response; LOB, limit of blank; TKI, tyrosine kinase inhibitors; pDNA, plasmid DNA; IS, international scale; EAC, Europe Against Cancer; FPR, false positivity rate; RT-qPCR, reverse-transcription quantitative PCR; qPCR, quantitative PCR; dPCR, digital PCR; LOD, limit of detection
• ISSN: 2352-5517; 2352-5517
• DOI: 10.1016/j.plabm.2021.e00210

BCR-ABL1 molecular detection using quantitative PCR (qPCR) methods is the golden standard of chronic myeloid leukemia (CML) monitoring. However, due to variable sensitivity of qPCR assays across laboratories, alternative methods are tested. Digital PCR (dPCR) has been suggested as a robust and reproducible option. Here we present a comparison of droplet dPCR with routinely used reverse-transcription qPCR (RT-qPCR) and automated GeneXpert systems. Detection limit of dPCR was above 3 BCR-ABL1 copies, although due to background amplification the resulting sensitivity was 0.01% BCR-ABL1 (MR4.0). Nevertheless, in comparison with GeneXpert, dPCR categorized more than 50% of the patients into different MR groups, showing a potential for improved BCR-ABL1 detection. •dPCR assays attain MR4.0 sensitivity due to a blank amplification 3 BCR-ABL1 copies.•Quantification principle of RT-qPCR and dPCR introduces bias in copy numbers.•dPCR provides more sensitive BCR-ABL1 measurement in >50% MR3.0-MR4.5 CML patients.