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Droplet Digital PCR Shows Lower Limits of Detection, Quantification for BCR-ABL Testing

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Researchers from Northwestern University and the University of Chicago have demonstrated in a new study that droplet-based digital PCR offers the ability to detect BCR-ABL1 fusion transcripts with a lower limit of detection and quantification than currently used quantitative PCR methods.

Specifically, the team used BioRad's QX100 Droplet Digital PCR platform with larger-than-recommended RNA inputs and pooling of wells to achieve limits of detection and quantification orders of magnitude lower than currently used real time quantitative PCR-based BCR-ABL1 testing.

A paper describing the work appeared online this month in the Journal of Molecular Diagnostics.

Lawrence Jennings, the study's first author and professor and laboratory director at Northwestern's Feinberg School of Medicine, told PCR Insider this week that he believes that the advantages of droplet digital PCR, regardless of platform, will make it the preferred technique for quantification of BCR-ABL1 fusion transcripts and other clinical targets across many labs.

In the study, Jennings and his colleagues used BioRad's QX100 system, but he said that this was because when his lab adopted the platform, newer competitors like RainDance had not yet entered the market.

"That decision wasn't based on advantages," he said. "And I would imagine RainDance, from the data out there, might even have an advantage now for [something like this], because they have more data points, assuming the background is as clean as we got."

According to Jennings, though the clinical applications for increasingly lower limits of detection and quantification are currently limited, they are growing.

Droplet digital PCR methods "have [mostly] been useful in the research realm to this point, in identifying rare events or quantifying copy number very precisely. But there is a huge potential in the clinical labs, which is why we wanted to put this paper out," he said.

Specifically, the study authors cite the fact that lower detection and quantification limits than can currently be achieved with standard RT-qPCR may be necessary in BCR-ABL1 transcript testing to help inform the management of leukemia patients who are highly responsive to tyrosine kinase inhibitors and might be eligible for cessation of therapy.

In their JMD report, Jennings and his co-authors describe a ddPCR method for detecting and quantifying BCR-ABL1 fusion transcripts at a limit of detection down to 0.001 percent on the International Scale. They developed and optimized their method using a strategy of larger-than-recommended RNA inputs per well and pooling of wells to increase the number of data points.

The group tested the method, pushing it to its lowest limits of detection by evaluating serially diluted samples.

Detection and quantification limits in digital PCR are constrained by three main factors, Jennings explained: first, the number of data points; secondly the amount of background one might have using a particular platform or instrument and particular set of probes and primers for an assay; and third, the amount of template loaded in.

"If you only have 20ng of RNA you are not going to get to a single copy in 10,000 cells as your limit of detection," he said.

In the study, the researchers determined that they could add up to 600 ng of total RNA to a single well without affecting accuracy. This, coupled with a strategy of combining the counts from multiple replicate wells, allowed the method to reach limits of detection orders of magnitude lower than what is achievable with current standard RT-qPCR testing.

According to Jennings, given the fact that the group's BCR-ABL1 assay had complete negative background — all the negative samples were entirely negative after PCR — higher RNA concentrations and/or more wells per sample would theoretically allow one to dramatically extend these limits using larger and larger sample inputs.

"Our background was very clean, so we could trust even a single positive droplet," he said. "So theoretically it could be unlimited, but to go another order of magnitude lower would require ten times the amount of RNA. Already we were putting 2.5 micrograms in; 25 would really be a lot but you could conceivably go lower. It would take half a plate, but you could go lower."

Jennings said his group has so far evaluated assays for the P210 and P190 forms of BCR-ABL, as well as developed at least one patient-specific set of probes and primers, with plans to do more.

The team has also worked with ddPCR to measure chimerism and low-level mosaicism, he said.

Digital PCR, Jennings explained, presents additional advantages for new test development over RT-qPCR, because it relies on endpoint detection, and the efficiency of amplification is less of a concern than it is in RT-qPCR, so developing and validating a new assay requires less work.

According to Jennings, other colleagues in his field are also testing BioRad and RainDance's droplet digital platforms for development of clinical laboratory tests. "I have no doubt that we will see more and more labs adapt a digital platform because the data is just better," he said.

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