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Digital PCR Reference Method Improves Inter-Lab qPCR Variability, Study Finds

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NEW YORK – Validating the quality of molecular diagnostics is difficult when standardized control materials are unavailable, yet determining whether the absolute quantity of a pathogen is clinically relevant requires being able to precisely determine its quantity. Now, a global team of lab measurement experts has drawn on the precision of digital PCR to show that a so-called reference measurement procedure (RMP) can harmonize nucleic acid amplification-based diagnostic test results between labs and potentially be used to perform quality assurance in the absence of physical control materials.

Material controls, like well-characterized and quantified pathogen DNA or RNA, can be used to generate calibration curves that allow labs to compute the starting amounts of nucleic acid in samples and compare results as part of external quality assurance, or EQA.

However, "the materials needed for EQA take time to standardize and cannot be distributed as quickly as the NAAT tests they were meant to support," an editorial published this week in Clinical Chemistry noted.

Indeed, while the technological capabilities of nucleic acid amplification testing have burgeoned over the past few decades, "there's not a big budget to develop reference materials," said Jim Huggett, a researcher at the University of Surrey and director of biological metrology at the National Measurement Laboratory at LGC. As a result, "standardization is an afterthought."

The problem is most common with emerging pathogens, like COVID, but it can also crop up in cases where new mutations cause dropouts or test escape, such as with a variant of chlamydia that evaded detection in Scandinavia.

In some cases, the lack of well-characterized and quantified DNA or RNA can be a major hurdle to widespread testing. "Rapidly producing external QA materials and methods is critical when dealing with new and emerging targets," said Peter Vallone, the applied genetics group leader at the US National Institute of Standards and Technology and a coauthor on the study. These materials, he said, "need to coincide with the development of commercial tests to ensure accuracy and confidence in the downstream measurements."

Access to inactivated SARS-CoV-2 virus, for example, hampered diagnostic development in the early days of the COVID-19 pandemic, and without standards, it was unclear how to interpret the 10,000-fold differences in limits of detection of early COVID tests. A 1,000-fold inter-lab variability also limited the usefulness of cycle threshold values as a proxy for viral load. Essentially, to say that there is a clinical consequence to results above or below a certain Ct implies a quantification that must in turn be calibrated and standardized between labs.

Huggett, who previously helped to craft the MIQE guidelines aimed at improving qPCR reproducibility, said that standardization is an important safeguard for molecular diagnostic test results that require immediate clinical intervention. In addition, knowing the accuracy of diagnostic testing can help governments globally to run simulations and forge their pandemic responses.

"We cannot know whether a method is accurate if it is not well standardized, and a method cannot be deemed critical if it is performing with unknown accuracy," he said.

Huggett's team previously showed that dPCR could be used as a RMP for HIV diagnostics, essentially enabling measurements to be linked to international standards through a series of calibrations and comparisons. The current work shows how dPCR can be used to support EQA value assignment over time, Huggett said, as labs can now "have a much better idea of the analyte concentration being used to assess the method."

Expanding on other earlier studies, Huggett and other international researchers specializing in lab standardization showed last month in Clinical Chemistry that digital PCR can provide absolute quantification that can in turn be used as the basis for EQA.

In the study, standards labs in the UK, US, and Germany deployed RT-dPCR to determine the absolute quantity of SARS-CoV-2 virus matched sets of samples. Their protocols were slightly different, but the method showed only a twofold difference in the range of results. On the other hand, while the median results using RT-qPCR agreed, they had between 10- and 50-fold differences in quantification.

Overall, the RT-dPCR approach offers "a paradigm shift in methodological agreement, given the orders of magnitude of variability observed in quantification using qPCR," the study authors concluded.

Megan Cleveland, a study coauthor and research biologist at NIST, said her team performed the digital measurements in replicates between June 2020 through November 2021, then returned the data to LGC for statistical analysis.

The NIST team has developed other RMPs in the past, Vallone also said, adding that these were previously known as definitive methods. Listed in a database maintained by the Joint Committee for Traceability in Laboratory Medicine, or JCTLM, these include RMPs for hormones, vitamin D metabolites, and other analytes. The team also collaborated on a review of dPCR as a method to characterize reference materials themselves.

While the NIST team also makes and distributes physical reference materials — for example a synthetic H5N1 material to validate bird flu assays — Vallone noted that its reference methods work doesn’t conflict with that role.

"They each have their place depending on the specific measurements and resources available, [and] either strategy can achieve the final goal of accuracy and confidence in the measurement," he said.

Huggett also noted that reference methods could take some of the burden off the material controls in the early days of an emerging pathogen, as "it's difficult for a single provider to calibrate the world."

A more rigorous future

For Huggett, there are notable national and regional discrepancies in uptake of approved, commercially available molecular diagnostics, as well as a "huge gap between what is promised from research and what actually impacts patients."

Standards, including material and methodological ones, can "play an important role in improving this situation and providing greater access to cutting-edge diagnostic technology," he believes.

The growth of digital PCR can also help here, Huggett suggested, having increased of late to four major players in the US and Europe, not including startups and dPCR instrument developers in China. 

"This is now an established technology," Huggett argued.

For the authors of the Clin Chem editorial, it is now time to consider the broader role of dPCR in infectious diseases, even beyond EQA approaches for global standardization of qPCR.

Challenges to broader uptake of dPCR include lack of automation, difficulties with sample management from the extraction step through to results reporting, and issues with the user-friendliness of the accompanying software, the authors said. Cost is also a big factor, they said, making dPCR less attractive than more budget-friendly qPCR, particularly in cases where "the need for quantification is relative and not necessarily related to the infectious status of the patient," they said.

Huggett also suggested that advances in biosensor-based detection are also lowering the limit of detection in molecular testing, while technologies like DNA printers can help provide material controls more quickly.

He is also involved in a Nucleic Acid Analysis Working Group (NAWG) at the International Committee for Weights and Measures that brings metrology institutes from across the world together and said that team is also exploring how dPCR can be performed as a high accuracy, SI traceable method, for example, with its recent work to standardize HER2 measurement.

"Standardized protocols empower the science," Huggett said, adding that hopefully now with the dPCR protocol, "whole procedures can be reproducible over time and between laboratories over a wide dynamic range of concentrations."