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At Threshold of Detection, False-Positive Droplets a Nuisance in ddPCR

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This article has been updated from a previous version to include comments from Bio-Rad.

Quantifying HIV levels in patients on antiviral therapy demands assays able to function well in the lowest ranges. A recent study in PLOS One has shown seminested qPCR to be more reliable for detecting ultra-low levels of cell-associated HIV RNA than droplet digital PCR, in part due to fluorescence in no-template control droplets.

According to author Ward De Spiegelaere, a researcher at the University of Ghent in Belgium, measurement of cell-associated HIV RNA could potentially reveal transcriptional activity of HIV that is not necessarily leading to production of virus, but which might show that the HIV reservoir is in some way active. His study quantified HIV RNA in peripheral blood mononuclear cells from 34 patients both on and off ART.

De Spiegelaere collaborated with Alexander Pasternak at The University of Amsterdam, who had developed a seminested qPCR assay for HIV RNA, to determine which protocol would be superior. He said that the two methods were similarly accurate, but ddPCR had occasional false-positive results. This problem seemed to crop up at random. "We have assays where we do 10 no-template controls and we have [all] non-positives, and others where we have two or three positives, it's quite stochastic," he said.

The two procedures ultimately take the same amount of time, De Spiegelaere said, but ddPCR is much less complicated to interpret, which makes the drawback of the false positives "a little bit of a bummer," he said.

The group used Bio-Rad's QX100 ddPCR platform, but now plans to repeat the study in collaboration with colleagues who have a Life Technologies system. However, De Spiegelaere said, "I already hear from them the throughput is much lower and the cost per assay is much higher, so it will have to be technically really superior to be better than the Bio-Rad system."

There have been a few other reports mentioning false-positive droplets in ddPCR at low ranges of detection, De Spiegelaere said. A seminal HIV DNA study in PLOS One by Matthew Strain at the University of California, San Diego, showed increased precision of ddPCR over qPCR on a set of 300 patient samples. That work also retrospectively analyzed over 500 no-template control wells and found false-positive events to occur infrequently but regularly, averaging between 0.1 to 0.4 events per well. This group suggested that the false-positive rate seemed to fluctuate over a period of several weeks. Since these events were not distinguishable from true positives, they claimed that this precluded arbitrary improvement of the limit of detection, which in their case was from 0.7 to 3 copies per million cells.

In a presentation this week at Cambridge Healthtech Institute's Molecular Medicine Tri-Conference in San Francisco, Strain stated definitively that real-time PCR is "not good enough" for the detection of residual HIV nucleic acids in patient samples, and provided additional evidence generated in his lab that droplet digital PCR is an ideal modality for this application. However, he also that the false positive events, or "noise," remain a limitation of the platform.

De Spiegelaere also referred to an Analytical Chemistry paper by Leonardo Pinheiro at the National Measurement Institute of Australia, which showed a low rate of fluorescence in no-template controls. Pinheiro confirmed this in an email with PCR Insider, but said the group hasn't done any further investigation since its publication. "My personal opinion is that the background signal reported as false positive can only be derived from formation of non-specific products or to be procedure related, i.e. possible contamination from materials, reagents, or the environment," he wrote. He added that he believed, "provided carefully optimized conditions ... digital PCR should deliver better accuracy and sensitivity than qPCR."

De Spiegelaere concurred in support of ddPCR, and said his lab is still using the Bio-Rad platform for its assays. "We're still doing it, and I would still advise people to do it, but taking into account the kind of gray zone in which you're not really sure." He said for sensitivity assays, detecting "whether there is anything at all, a regular PCR would still be best, because you can sequence your template afterwards." However, "if you want to do quantification, go for digital," he advised, "because the limits where you start to see false positives is in the limit where you actually have stochastic sampling error."

For higher ranges of detection, these false positives are also not an issue in De Spiegelaere's lab. "We are doing ddPCR, for example, for total genomic DNA regularly, and for HIV DNA, and they are perfect, they are very accurate, and in that range they are really superior," De Spiegelaere said.

Bio-Rad is aware of the issue. Indeed, two authors on the Pinheiro paper were affiliated with Bio-Rad.

In addition, in an email to PCR Insider, George Karlin-Neumann, director of scientific affairs at Bio-Rad's Digital Biology Center, noted that the company is "reaching out to [De Spiegelaere's lab] to better understand their situation, but based on what's been said in the study ... in our experience, assay re-design and running conditions could potentially eliminate such sporadic false positives. We know that under optimal conditions the technology is capable of providing sensitivity beyond one in 1 million (one target in a background of 1 million non-target genomes) in both our hands and those of our customers."

As for the cause of the false positives, De Spiegelaere doesn't think it is contamination. "We made some PCRs that cannot work, with only forward primers or only reverse primers, so normally nothing should happen if there would be contamination," he said. He hypothesized self-hydrolyzing of the probe may be the culprit, and also noted that the frequency of the problem seems to be assay-dependent, suggesting perhaps issues with particular probe batches.

In the meantime, De Spiegelaere continues to use the platform and said, "I personally believe that the Bio-Rad [platform] is just the first generation of its kind, and I really hope that they can solve the false-positive issue."

Bio-Rad's Karlin-Neumann noted that "we make available to our customers a suite of ddPCR wet lab-validated assays to provide optimal performance in their hands, and also welcome partnerships with our users to better understand areas where the technology can be made still more robust and serviceable."

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