The performance of human cytomegalovirus digital PCR reference measurement procedure in seven external quality assessment schemes over four years.

A candidate digital PCR (dPCR)-based reference measurement procedure for quantification of human cytomegalovirus (hCMV) was evaluated in 10 viral load comparison schemes (seven external quality assessment (EQA) and three additional training schemes) organized by INSTAND e.V. over four years (between September 2014 and March 2018). Four metrology institutes participated in these schemes using the same extraction method and dPCR measurement procedure for the hCMV specific target sequence of UL54 gene. The calibration independent reference measurement procedure results from the metrology institutes were compared to the results of the clinical diagnostic laboratories applying hCMV qPCR measurement procedures calibrated to reference materials. While the criteria for the acceptable deviation from the target value interval for INSTAND's EQA schemes is from -0.8 log10 to +0.8 log10, the majority of dPCR results were between -0.2 log10 to +0.2 log10. Only 4 out of 45 results exceeded this interval with the maximum deviation of -0.542 log10. In the training schemes containing samples with lower hCMV concentrations, more than half of the results deviated less than ±0.2 log10 from the target value, while more than 95% deviated less than ±0.4 log10 from the target value. Evaluation of intra- and inter-laboratory variation of dPCR results confirmed high reproducibility and trueness of the method. This work demonstrates that dPCR has the potential to act as a calibration independent reference measurement procedure for the value assignment of hCMV calibration and reference materials to support qPCR calibration as well as ultimately for routine hCMV load testing.

Assessment of Digital PCR as a Primary Reference Measurement Procedure to Support Advances in Precision Medicine.

BACKGROUND: Genetic testing of tumor tissue and circulating cell-free DNA for somatic variants guides patient treatment of many cancers. Such measurements will be fundamental in the future support of precision medicine. However, there are currently no primary reference measurement procedures available for nucleic acid quantification that would support translation of tests for circulating tumor DNA into routine use. METHODS: We assessed the accuracy of digital PCR (dPCR) for copy number quantification of a frequently occurring single-nucleotide variant in colorectal cancer (KRAS c.35G>A, p.Gly12Asp, from hereon termed G12D) by evaluating potential sources of uncertainty that influence dPCR measurement. RESULTS: Concentration values for samples of KRAS G12D and wild-type plasmid templates varied by <1.2-fold when measured using 5 different assays with varying detection chemistry (hydrolysis, scorpion probes, and intercalating dyes) and <1.3-fold with 4 commercial dPCR platforms. Measurement trueness of a selected dPCR assay and platform was validated by comparison with an orthogonal method (inductively coupled plasma mass spectrometry). The candidate dPCR reference measurement procedure showed linear quantification over a wide range of copies per reaction and high repeatability and interlaboratory reproducibility (CV, 2%-8% and 5%-10%, respectively). CONCLUSIONS: This work validates dPCR as an SI-traceable reference measurement procedure based on enumeration and demonstrates how it can be applied for assignment of copy number concentration and fractional abundance values to DNA reference materials in an aqueous solution. High-accuracy measurements using dPCR will support the implementation and traceable standardization of molecular diagnostic procedures needed for advancements in precision medicine.

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

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.

Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA.

Quantitative PCR (qPCR) is an important tool in pathogen detection. However, the use of different qPCR components, calibration materials and DNA extraction methods reduces comparability between laboratories, which can result in false diagnosis and discrepancies in patient care. The wider establishment of a metrological framework for nucleic acid tests could improve the degree of standardisation of pathogen detection and the quantification methods applied in the clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures, and to facilitate characterisation of suitable certified reference materials. Digital PCR (dPCR) has already been used for pathogen quantification by analysing nucleic acids. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessment of its actual performance characteristics is needed before it can be implemented in a metrological framework, and to allow adequate estimation of measurement uncertainties. Here, four laboratories demonstrated reproducibility (expanded measurement uncertainties below 15%) of dPCR for quantification of DNA from human cytomegalovirus, with no calibration to a common reference material. Using whole-virus material and extracted DNA, an intermediate precision (coefficients of variation below 25%) between three consecutive experiments was noted. Furthermore, discrepancies in estimated mean DNA copy number concentrations between laboratories were less than twofold, with DNA extraction as the main source of variability. These data demonstrate that dPCR offers a repeatable and reproducible method for quantification of viral DNA, and due to its satisfactory performance should be considered as candidate for reference methods for implementation in a metrological framework.

The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis.

BACKGROUND: Real-time PCR (qPCR) based methods, such as the Xpert MTB/RIF, are increasingly being used to diagnose tuberculosis (TB). While qualitative methods are adequate for diagnosis, the therapeutic monitoring of TB patients requires quantitative methods currently performed using smear microscopy. The potential use of quantitative molecular measurements for therapeutic monitoring has been investigated but findings have been variable and inconclusive. The lack of an adequate reference method and reference materials is a barrier to understanding the source of such disagreement. Digital PCR (dPCR) offers the potential for an accurate method for quantification of specific DNA sequences in reference materials which can be used to evaluate quantitative molecular methods for TB treatment monitoring. METHODS: To assess a novel approach for the development of quality assurance materials we used dPCR to quantify specific DNA sequences in a range of prototype reference materials and evaluated accuracy between different laboratories and instruments. The materials were then also used to evaluate the quantitative performance of qPCR and Xpert MTB/RIF in eight clinical testing laboratories. RESULTS: dPCR was found to provide results in good agreement with the other methods tested and to be highly reproducible between laboratories without calibration even when using different instruments. When the reference materials were analysed with qPCR and Xpert MTB/RIF by clinical laboratories, all laboratories were able to correctly rank the reference materials according to concentration, however there was a marked difference in the measured magnitude. CONCLUSIONS: TB is a disease where the quantification of the pathogen could lead to better patient management and qPCR methods offer the potential to rapidly perform such analysis. However, our findings suggest that when precisely characterised materials are used to evaluate qPCR methods, the measurement result variation is too high to determine whether molecular quantification of Mycobacterium tuberculosis would provide a clinically useful readout. The methods described in this study provide a means by which the technical performance of quantitative molecular methods can be evaluated independently of clinical variability to improve accuracy of measurement results. These will assist in ultimately increasing the likelihood that such approaches could be used to improve patient management of TB.

International Comparison of Enumeration-Based Quantification of DNA Copy-Concentration Using Flow Cytometric Counting and Digital Polymerase Chain Reaction.

Enumeration-based determination of DNA copy-concentration was assessed through an international comparison among national metrology institutes (NMIs) and designated institutes (DIs). Enumeration-based quantification does not require a calibration standard thereby providing a route to "absolute quantification", which offers the potential for reliable value assignments of DNA reference materials, and International System of Units (SI) traceability to copy number 1 through accurate counting. In this study, 2 enumeration-based methods, flow cytometric (FCM) counting and the digital polymerase chain reaction (dPCR), were compared to quantify a solution of the pBR322 plasmid at a concentration of several thousand copies per microliter. In addition, 2 orthogonal chemical-analysis methods based on nucleotide quantification, isotope-dilution mass spectrometry (IDMS) and capillary electrophoresis (CE) were applied to quantify a more concentrated solution of the plasmid. Although 9 dPCR results from 8 laboratories showed some dispersion (relative standard deviation [RSD] = 11.8%), their means were closely aligned with those of the FCM-based counting method and the orthogonal chemical-analysis methods, corrected for gravimetric dilution factors. Using the means of dPCR results, the RSD of all 4 methods was 1.8%, which strongly supported the validity of the recent enumeration approaches. Despite a good overall agreement, the individual dPCR results were not sufficiently covered by the reported measurement uncertainties. These findings suggest that some laboratories may not have considered all factors contributing to the measurement uncertainty of dPCR, and further investigation of this possibility is warranted.

Assessment of the real-time PCR and different digital PCR platforms for DNA quantification.

Digital PCR (dPCR) is beginning to supersede real-time PCR (qPCR) for quantification of nucleic acids in many different applications. Several analytical properties of the two most commonly used dPCR platforms, namely the QX100 system (Bio-Rad) and the 12.765 array of the Biomark system (Fluidigm), have already been evaluated and compared with those of qPCR. However, to the best of our knowledge, direct comparison between the three of these platforms using the same DNA material has not been done, and the 37 K array on the Biomark system has also not been evaluated in terms of linearity, analytical sensitivity and limit of quantification. Here, a first assessment of qPCR, the QX100 system and both arrays of the Biomark system was performed with plasmid and genomic DNA from human cytomegalovirus. With use of PCR components that alter the efficiency of qPCR, each dPCR platform demonstrated consistent copy-number estimations, which indicates the high resilience of dPCR. Two approaches, one considering the total reaction volume and the other considering the effective reaction size, were used to assess linearity, analytical sensitivity and variability. When the total reaction volume was considered, the best performance was observed with qPCR, followed by the QX100 system and the Biomark system. In contrast, when the effective reaction size was considered, all three platforms showed almost equal limits of detection and variability. Although dPCR might not always be more appropriate than qPCR for quantification of low copy numbers, dPCR is a suitable method for robust and reproducible quantification of viral DNA, and a promising technology for the higher-order reference measurement method.

Digital PCR for direct quantification of viruses without DNA extraction.

DNA extraction before amplification is considered an essential step for quantification of viral DNA using real-time PCR (qPCR). However, this can directly affect the final measurements due to variable DNA yields and removal of inhibitors, which leads to increased inter-laboratory variability of qPCR measurements and reduced agreement on viral loads. Digital PCR (dPCR) might be an advantageous methodology for the measurement of virus concentrations, as it does not depend on any calibration material and it has higher tolerance to inhibitors. DNA quantification without an extraction step (i.e. direct quantification) was performed here using dPCR and two different human cytomegalovirus whole-virus materials. Two dPCR platforms were used for this direct quantification of the viral DNA, and these were compared with quantification of the extracted viral DNA in terms of yield and variability. Direct quantification of both whole-virus materials present in simple matrices like cell lysate or Tris-HCl buffer provided repeatable measurements of virus concentrations that were probably in closer agreement with the actual viral load than when estimated through quantification of the extracted DNA. Direct dPCR quantification of other viruses, reference materials and clinically relevant matrices is now needed to show the full versatility of this very promising and cost-efficient development in virus quantification.

Standardization of Nucleic Acid Tests for Clinical Measurements of Bacteria and Viruses.

Nucleic acid-based tests for infectious diseases currently used in the clinical laboratory and in point-of-care devices are diverse. Measurement challenges associated with standardization of quantitative viral load testing are discussed in relation to human cytomegalovirus, BK virus, and Epstein-Barr virus, while the importance of defining the performance of qualitative methods is illustrated with Mycobacterium tuberculosis and influenza virus. The development of certified reference materials whose values are traceable to higher-order standards and reference measurement procedures, using, for instance, digital PCR, will further contribute to the understanding of analytical performance characteristics and promote clinical data comparability.

Optimising droplet digital PCR analysis approaches for detection and quantification of bacteria: a case study of fire blight and potato brown rot.

Here we report on the first assessment of droplet digital PCR (ddPCR) for detection and absolute quantification of two quarantine plant pathogenic bacteria that infect many species of the Rosaceae and Solanaceae families: Erwinia amylovora and Ralstonia solanacearum. An open-source R script was written for the ddPCR data analysis. Analysis of a set of samples with known health status aided the assessment and selection of different threshold settings (QuantaSoft analysis, definetherain pipeline and manual threshold), which led to optimal diagnostic specificity. The interpretation of the E. amylovora ddPCR was straightforward, and the analysis approach had little influence on the final results and the concentrations determined. The sensitivity and linear range were similar to those for real-time PCR (qPCR), for the analysis of both bacterial suspensions and plant material, making ddPCR a viable choice when both detection and quantification are desired. With the R. solanacearum ddPCR, the use of a high global threshold was necessary to exclude false-positive reactions that are sometimes observed in healthy plant material. ddPCR significantly improved the analytical sensitivity over that of qPCR, and improved the detection of low concentrations of R. solanacearum in potato tuber samples. Accurate and rapid absolute quantification of both of these bacteria in pure culture was achieved by direct ddPCR. Our data confirm the suitability of these ddPCR assays for routine detection and quantification of plant pathogens and for preparation of defined in-house reference materials with known target concentrations.