Does dead volume impact dPCR sensitivity?
Here’s a digital PCR (dPCR) topic that is quite alive in papers, forums and coffee chats: dead volume. But unlike ghosts, zombies or pets from Stephen King novels, this deadly term shouldn’t scare anyone from dPCR.
Here’s why there’s nothing to be afraid of. Dead volume, or void volume, is easy to understand, its effects on dPCR sensitivity have been investigated and data from studies already exists. Instead of avoiding void volume, let’s explore it together.
What is dead volume in dPCR?
Dead volume, or void volume, is the volume of dPCR reaction that is prepared but not actually included in the partitioning and analysis of the dPCR reaction. Theoretically, a lower dead volume could lead to higher sensitivity, since more of the loaded template is being analyzed. This could be important when measuring low concentrations of a target, for example, rare mutations. However, dead volume alone does not determine assay sensitivity. What matters is the total number of target molecules that ultimately enter the analyzed partition.
Is dead volume equal across dPCR platforms?
No. The way the dPCR reaction is partitioned and distributed in chambers or droplets might reflect in the dead volume of that method. Nanoplate dPCR can have larger void volume by design. However, droplets are fragile and could burst or fuse leading to higher and variable dead volume (which can also skew the analysis). Bursting or fusion is not an issue for the solid chambers of nanoplate dPCR.
Other PCR technologies may have lower dead volume, but they suffer from additional limitations, such as cost, workflow complications and throughput capabilities, that could limit their applicability.
How important is dead volume to dPCR sensitivity?
The most important factors in determining the relative sensitivity of dPCR systems are:
- Template addition volume – the total amount of template that can be added to each reaction for analysis – the more sample added to a reaction, the more template will be present for analysis
- Dead volume – the amount of the reaction that is ultimately analyzed by the dPCR instrument – risk that very rare target molecules remain in the dead volume and are not detected or that eluate aliquots from the same sample source result in vast differences in abundance of the target molecules
To truly consider the impact on sensitivity, we must take both factors into account. What happens if we combine them into one, calling it the template analyzed volume?
Let’s compare a QIAcuity Nanoplate 26K with a droplet digital PCR system with a dead volume of ~43 to 48% (with the assumption of a 15,000 droplet count and using measured droplet volumes of 0.718 nL and 0.786 nL) (Table 1).
Despite its higher unanalyzed volume, the QIAcuity Nanoplate 26K can accept up to 26 µL of sample and analyzes a higher template volume than a droplet-based system (Table 1). Consequently, QIAcuity analyzes substantially more sample-derived template molecules, thereby increasing the probability of detecting ultra-low-abundance targets. This is particularly important for rare mutation detection, liquid biopsy and viral detection applications.
If we define a rare event as a template at a concentration of ~0.1 to 0.4 copies/µL, this would mean ~3 to 20 copies in a 30 µL eluate. With Table 1 in mind, more copies are transferred and analyzed using the QIAcuity Digital PCR System compared to other technologies, despite their similar or lower non-analyzed volumes (Table 2).
What does published research have to say about dead volume in dPCR?
One paper shows that despite differences in dead volumes, platforms achieve statistically comparable limit of detection (LODs) for assays. Just a reminder, LOD refers to the lowest analyte concentration that can be distinguished from zero, with a specified level of confidence.
The authors speculate that the rate-limiting step in dPCR is the subsampling from the tube containing the template – a Poisson process – rather than the probability of the template being included in the effective dPCR reaction volume (3).
Interestingly, platforms with lower dead volume have even been shown to display higher variability across different reactions than platforms with higher dead volume percentages. These authors also found no association between dead volume and the coefficient of variation across platforms at specific dilutions (4).
Another paper points out that even if you’re nervous about dead volume, larger volumes of DNA could be added to a reaction to obtain a larger effective concentration. This would have no impact on the LOD if copy numbers per total reaction volume were considered, but it would allow detection and quantification in samples with lower target concentrations (5).
This goes hand-in-hand with QIAGEN results (1,2) demonstrating that when you load more, you see more. Or in other words:
The number of target molecules analyzed, not the percentage of reaction volume analyzed, is the primary determinant of sensitivity in dPCR.
We hope we’ve put your dPCR dead volume questions to rest. But if not, how about a short video to wrap this up? Watch QIAgenius take you through this topic in just 3 minutes in a video about void volume in dPCR.
Summary: Should you be deadly concerned about dPCR dead volume?
No. Data and independent research show that dead volume is a manageable design characteristic, not a threat to assay sensitivity or data reliability. In fact:
- Sensitivity is determined by the total number of target molecules actually analyzed, rather than the percentage of unused reaction volume
- Platforms like the QIAcuity with the Nanoplate 26K accommodate a much larger template addition volume (~26 µL). This allows them to analyze more total sample volume (~14 µL) than droplet systems, increasing the probability of capturing ultra-low abundance targets (e.g., rare mutations)
- Published studies confirm that varying dead volumes do not negatively impact the statistical limit of detection (LOD) and that lower dead-volume platforms do not yield better consistency or lower reaction variability
References
- QIAGEN R&D. Internal data from experiments carried out in Hilden, Germany. 2026.
- Bussmann M et al. Impact of template addition volume and analyzed volume on digital PCR sensitivity. QIAGEN, 2022. https://www.qiagen.com/us/resources/protocols/prom-20715-001-1127766-an-dpcr-nanoplate-dead-vol-0622-ww
- Ghosh S, Bivins A. A head-to-head comparison of two microfluidic digital PCR platforms for fecal contamination testing in surface waters. ACS ES&T Water. 2025;6:429–437.
- Pitton M, et al. Performance and characteristics of three digital PCR platforms for detection and quantification of viral targets in wastewater. J Appl Microbiol. 2025;136(10):lxaf243.
- Jelenčić A, Stebih D, Demšar T, Dobnik D. Simple, fast, reliable: Multiplex digital PCR quantification of 19 genetically modified soybean events. GM Crops Food. 2026;17(1):2635816.