NEW YORK – Now a quarter-century-old technology, digital PCR in 2023 continued its push into clinical applications, as system developers launched new instruments and assays designed to expand dPCR's utility in clinical labs.
Meanwhile, in research use markets, funding challenges temporarily stalled dPCR uptake in biopharma, while new growth in wastewater testing augured continued expansion of the technology.
Digital PCR is generally considered more precise and inhibitor-resistant than qPCR with a lower cost than sequencing, and these unique features have made it attractive to end users in many different domains. The number of commercially available digital PCR systems has doubled in the past three years. Flagship instruments — the QX systems from Bio-Rad Laboratories, the Naica from Stilla Technologies, and the QuantStudio 3D that Thermo Fisher Scientific acquired from Life Technologies — have now been joined by Thermo Fisher's Absolute Q, Qiagen's QiAcuity, and Roche Diagnostics' Digital LightCycler. Newcomer Enumerix is also developing a system using a method called UltraPCR, while Bio-Rad has been working on an instrument using continuous-flow digital PCR technology that it acquired with startup Dropworks.
All of these systems differ in the way that the PCR reaction droplets are formed, whether they physically partition droplets on plates or arrays or suspend them in tubes, as well as in the number of instruments required to perform an experiment, the number of optical channels, and potential for multiplexing. They also differ in overall workflow, time to results, and instrument and consumables cost.
Bio-Rad recently acknowledged the intensifying competitive pressures in the digital PCR space in its third quarter earnings call in October, with Chief Operating Officer Andrew Last stating that some competitors are "playing more in the lower-end segments," which Bio-Rad also plans to target with the QX Continuum platform in 2024.
While the overall trend in newly launched dPCR systems has been for an increasing number of optical channels and multiplexing capabilities, developers seem to now also be targeting clinical end users with simplified, single-instrument workflows that allow for varying throughputs.
For example, in November Stilla launched the Nio+ dPCR system. It sports seven optical channels, adding a low Stokes-shift dye channel to a set of six channels that is becoming more common in the dPCR space. The Nio+ also supports a new method using color combinations to multiplex up to 21 targets. But the system was also designed to be user-friendly, consisting of one benchtop instrument with a large touchscreen interface and featuring random access capabilities and continuous loading of experiments. It also has modifiable throughput, processing as many as 48 of the firm's 16-well Ruby chips per day, or up to 768 samples per day.
On the occasion of the Nio+ launch, Wim Trypsteen, a cofounder of the Ghent University Digital PCR Consortium in Belgium, said that there are only a handful of single-instrument digital PCR systems currently available commercially but, in his opinion, this and other features focused on ease of use are becoming essential.
Prior to the launch of Nio+, Stilla had explicitly pivoted to clinical oncology applications earlier in 2023 with the introduction of its load-and-go Ruby chip. The firm also announced the launch of 20 new oncology assays for its Naica system codeveloped with ApexBio in addition to 10 kits the firm already developed with French liquid biopsy firm ID Solutions, and a partnership with Atila to codevelop up to 20 liquid biopsy assays.
Roche also specifically targeted the clinical space when it launched its single-instrument Digital LightCycler dPCR system in late 2022.
"Where we really think that digital PCR has its largest potential is moving into a clinical space to provide oncology and disease monitoring testing," said Miriam Fend, Roche's life cycle leader of PCR systems.
The Digital LightCycler features high sample input volume, six-color detection, and easier custom assay development. It also has an automated workflow that divides a sample into 100,000 partitions, yields results in 1.5 to 3 hours, uses intuitive software for data analysis and results reporting, and can be connected to LIS systems. Being a plate-based system also makes it simpler to use, Fend said, which can broaden access for labs that need more standardization and an easier workflow.
Mikael Kubista, founder and CEO of TATAA Biocenter, said at the time that a "unique and highly appreciated feature" of the new Roche dPCR system is its separated workflows for development and regulated bioanalyses.
"The development mode offers the flexibility we need when designing and validating custom assays for clients' pharmacodynamic and pharmacokinetic studies, while the regulated bioanalysis mode satisfies the requirements for good laboratory practice studies and enables efficient technology and method transfer when requested," he said.
Roche cited its focus on clinical labs, as well, when it launched its updated LightCycler PRO qPCR system in late 2023, which features seven optical channels as well as dedicated workflows enabling users to run research-use-only assays and IVD tests.
Also targeting the clinical space, Qiagen in 2023 outlined plans to pursue CE marking and US Food and Drug Administration clearance in parallel for a BCR-ABL assay on its QiAcuity system and said it expects its first sales of dPCR clinical tests in 2024. The firm said it will first pursue an oncology menu, but it will also consider infectious disease applications and possibly noninvasive prenatal testing applications.
Bio-Rad Laboratories had previously branched into clinical IVD testing for its QXDx system when it was cleared along with a BCR-ABL %IS Kit in 2019. It is also developing digital PCR diagnostics for reproductive health, infectious disease, and transplant monitoring while also focusing on biopharma customers, the firm said in 2022.
Thermo Fisher Scientific, meanwhile, launched its Absolute Q dPCR system in 2021 with sights set on the clinical space, research-use, and applied markets. In late 2022, the firm launched more than 50 research-use tests for the system, including 20 liquid biopsy tests, as well as a Digital PCR Custom Assay Design tool.
Although it is not a dPCR instrument maker, assay and reagent developer ChromaCode also strategically pivoted into the clinical dPCR space in 2023. The firm presented data at the American Association for Cancer Research meeting the prior year on the use of its HDPCR technology with the Absolute Q dPCR system, showing its NSCLC assay had a limit of detection as low as 10 variant copies in a background of 10,000 haploid human genome DNA copies.
ChromaCode launched the NSCLC assay in July 2023, and in November it presented data at the Association for Molecular Pathology meeting showing 92 percent and 99 percent specificity for the HDPCR assay compared to a sequencing-based test. Of note, ChromaCode and Bio-Rad are also currently involved in patent litigation, with ChromaCode in October filing a lawsuit against Bio-Rad for allegedly infringing patents related to dPCR multiplexing. In December that lawsuit was ordered to alternative dispute resolution in the US District Court for the Northern District of California.
Digital in the clinic
In addition to new clinical products from dPCR developers, 2023 also saw a slew of clinical trial results and uptake of these systems for clinical use.
In the oncology space, Geneoscopy achieved a milestone with the publication of positive results from its colorectal cancer clinical trial using the Bio-Rad QXDx system. Hummingbird Diagnostics said it is developing a version of its 18-miRNA signature miLung lung cancer test that uses digital PCR rather than NGS. The Mayo Clinic initiated a Phase II trial of Naveris' NavDx dPCR test to detect HPV-driven head and neck cancer, while GenoMe Diagnostics disclosed that it is prepping a dPCR-based ovarian cancer test for submission in the US, the UK, and the EU. Meanwhile, a Cedars-Sinai research team also described a prostate cancer status test using dPCR.
In October, Qiagen partnered with Myriad to develop companion diagnostic tests using next-generation sequencing workflows and the QiAcuity digital PCR platform. The firms said the goal was to provide both lab-developed oncology tests for the US clinical market and distributable companion diagnostic test kits for the global market. They will initially focus on developing assays in collaboration with pharmaceutical partners.
In other clinical testing, Oncocyte received Medicare coverage for a kidney transplant rejection test using Bio-Rad's Droplet Digital PCR, and after obtaining private financing earlier in 2023, Cardio Diagnostics inked a distribution agreement in India for a test that uses six methylation-sensitive dPCR assays and 10 SNPs to determine coronary heart disease status.
Bio-Rad's QX One dPCR system was also selected for use in spinal muscular atrophy screening in Hong Kong. A team of researchers in that country is also adapting a cell-free DNA-based preeclampsia test to the QX One instrument, noting that a ddPCR-based test is more than 30 times less expensive than long-read sequencing.
Finally, 2023 also saw some growth in noninvasive prenatal testing as an application space for dPCR.
Enumerix emerged from stealth mode in December 2022 with a system that could generate 30 million droplets per reaction. The firm, which was founded by pioneers in NIPT, described it as an early target market, and has developed an NIPT assay that detects a total of 222 loci in chromosomes 13, 18, and 21 in a single-tube workflow.
And, in research published in Scientific Reports in December 2023, Czech Republic scientists used a multiplex dPCR approach and Stilla's dPCR system for trisomy 21 testing. The dPCR-based screening test was in complete agreement with the results of a more invasive diagnostic procedure, with 100 percent sensitivity, specificity, and positive and negative predictive values. The approach was also cheaper than other methods, with faster analysis speed, which the authors concluded makes it a potential screening method for implementation into the clinical workflow.
Biopharma down, wastewater up
Among nonclinical applications for dPCR, the biopharma space has become an attractive niche for developers, and the technology has emerged as an ideal tool to help address challenges in developing and manufacturing cell and gene therapies.
In recent years, many dPCR developers have launched special kits catering to biopharma end users. For example, in 2023 Qiagen launched a customized cell and gene therapy dPCR assay design service as well as biopharma kits for cell and gene therapy quality control.
But 2023 also saw a tightening of funding available for biopharma research and development, which led to decreased purchasing, particularly for smaller biotech firms, and impacted some dPCR developers. Qiagen reportedly did not suffer large setbacks in this business, but Bio-Rad Laboratories lowered its guidance in May, August, and October, attributed in part to the softness in biopharma funding.
In initiating coverage of Bio-Rad in December, however, UBS investment analysts said that downturns and headwinds in digital PCR are product cycles that will likely be overcome.
Another major applied market use of dPCR is wastewater surveillance, also called wastewater-based epidemiology. The field of WBE was greatly accelerated by the pandemic, and the US Centers for Disease Control and Prevention established the National Wastewater Surveillance System in September 2020 to track SARS-CoV-2 in sewage.
The program began to transition from being sited primarily in academic and contract labs to public health labs in 2022. At that time, Amy Kirby, the director of the division, said dPCR "is the most sensitive approach to wastewater surveillance, and it is very robust to the inhibitors present in wastewater."
The CDC previously surveyed all wastewater testing labs in collaboration with the Association of Public Health Laboratories and found that 60 percent of public health labs already have digital PCR systems. About half have Qiagen and the other half Bio-Rad systems, although there are a few adopters of Thermo Fisher Scientific and other platforms.
The CDC said it plans to develop and validate its panels and protocols for use on both Qiagen and Bio-Rad instruments, noting that there are pros and cons to both systems.
The NWSS team subsequently added surveillance for mpox, formerly known as monkeypox, in 2022, at about 500 sites. This approach was shown to be able to detect only a handful of mpox cases in a community in a New England Journal of Medicine article.
In June 2023, the CDC announced it would greatly expand the number of targets it intends to track using dPCR panels. The new panels are being developed under a contract with GT Molecular, a Fort Collins, Colorado-based test developer, and ultimately will detect approximately two dozen pathogens of interest in wastewater using dPCR.
National wastewater surveillance was reportedly somewhat disrupted later in 2023, however.
In October, the CDC announced it had awarded a contract for wastewater testing at certain cites not yet surveilled by state and local public health labs to Verily Life Sciences, formerly known as Google Life Sciences. In its Q3 earnings call, Bio-Rad executives noted that Verily uses the firm's QX600 for wastewater testing.
However, the previous contract holder, Biobot Analytics, filed a protest over the awarding of the testing contract, and this appears to have disrupted testing for mpox specifically. The CDC's website currently states that testing for mpox has resumed for its approximately 350 commercial contract sites, noting that there had been a temporary delay since Sept. 15, 2023.