Droplet volume variability as a critical factor for accuracy of absolute quantification using droplet digital PCR

Abstract

Accurate and precise nucleic-acid quantification is crucial for clinical and diagnostic decisions, as overestimation or underestimation can lead to misguided treatment of a disease or incorrect labelling of the products. Digital PCR is one of the best tools for absolute nucleic-acid copy-number determination. However, digital PCR needs to be well characterised in terms of accuracy and sources of uncertainty. With droplet digital PCR, discrepancies between the droplet volume assigned by the manufacturer and measured by independent laboratories have already been shown in previous studies. In the present study, we report on the results of an inter-laboratory comparison of different methods for droplet volume determination that is based on optical microscopy imaging and is traceable to the International System of Units. This comparison was conducted on the same DNA material, with the examination of the influence of parameters such as droplet generators, supermixes, operators, inter-cartridge and intra-cartridge variability, and droplet measuring protocol. The mean droplet volume was measured using a QX200™ AutoDG™ Droplet Digital™ PCR system and two QX100™ Droplet Digital™ PCR systems. The data show significant volume differences between these two systems, as well as significant differences in volume when different supermixes are used. We also show that both of these droplet generator systems produce droplets with significantly lower droplet volumes (13.1%, 15.9%, respectively) than stated by the manufacturer and previously measured by other laboratories. This indicates that to ensure precise quantification, the droplet volumes should be assessed for each system.Electronic supplementary materialThe online version of this article (10.1007/s00216-017-0625-y) contains supplementary material, which is available to authorized users.

Critical assessment of digital PCR for the detection and quantification of genetically modified organisms

“…Discrepancies between partition volumes assigned by the manufacturer and measured in independent laboratories have been reported for ddPCR platforms [38, 58, 80–82]. At NIB, Bio-Rad’s QX100/QX200 platform has been tested most rigorously.…”

“…With a new line of cartridges, the software was updated to consider a volume of 0.85 nL. Nevertheless, measurement of the new cartridges showed a deviation from Bio-Rad’s default value, with volumes of 0.767 nL [81] and 0.715 nL [82]. The most recent study [82] not only showed that the volume of droplets is significantly lower but also that the volume of droplets is affected by type of super mix (for probes or EvaGreen) and the type of droplet generator (manual or automated).…”

“…Nevertheless, measurement of the new cartridges showed a deviation from Bio-Rad’s default value, with volumes of 0.767 nL [81] and 0.715 nL [82]. The most recent study [82] not only showed that the volume of droplets is significantly lower but also that the volume of droplets is affected by type of super mix (for probes or EvaGreen) and the type of droplet generator (manual or automated). More interestingly, when Bio-Rad’s default droplet volume was used at NIB, lower absolute target concentrations were determined by ddPCR (Bio-Rad) compared with cdPCR (Biomark HD) not only for GMO samples (unpublished data) but also for other samples, such as human cytomegalovirus [83].…”

“…More interestingly, when Bio-Rad’s default droplet volume was used at NIB, lower absolute target concentrations were determined by ddPCR (Bio-Rad) compared with cdPCR (Biomark HD) not only for GMO samples (unpublished data) but also for other samples, such as human cytomegalovirus [83]. The values obtained with the ddPCR and cdPCR platforms were much closer when correction-factor-based measurement was performed [82]. This phenomenon was also observed for the RainDrop platform.…”

“…The droplet volume was corrected on the basis of the droplet measurements, and the measured volume of 4.39 pL was used to calculate the target concentration instead of 5 pL provided by the manufacturer [58]. As concluded by Bogožalec Košir et al [82], it is of great importance to know the exact droplet volume, which might even be laboratory specific.…”