Reporter emission multiplexing in digital PCRs (REM-dPCRs): direct quantification of multiple target sequences per detection channel by population specific reporters.

Digital PCRs (dPCRs) are widely used methods for the detection and quantification of rare abundant sequences relevant to fields such as liquid biopsy or oncology. In order to increase the information content and save valuable sample materials, there is a significant need for digital multiplexing methods that are easy to establish, analyse, and interpret, and ideally allow the usage of existing lab equipment. Herein, we present a novel reporter emission multiplexing approach for the digital PCR method (REM-dPCR), which meets these requirements. It further increases the multiplexing capacity of commercial dPCR devices. For example, we present a stepwise increase in multiplexing degrees from a monochrome two-plex assay in one detection channel to a six-plex REM-dPCR assay in a three-color dPCR device for KRAS/BRAF single nucleotide polymorphism (SNP) target sequences. The guidelines for the REM-dPCR design are presented, and the process from duplex to six-plex assay establishment, taking into account the target sequence-dependent effects on assay performance, is discussed. Furthermore, the assay-specific, sensitive and precise quantification of different fractions of KRAS mutant and wild-type DNA sequences in different ratios is demonstrated. To increase the device capacitance and the degree of multiplexing, the REM-dPCR uses the advantage of n target-independent reporter molecules in combination with target sequence-specific mediator probes. Different reporter types are labelled with fluorophores of different signal intensities but not necessarily different emission spectra. This leads to the generation of n independent single-positive populations in the dataspace, created by k detection channels, whereby n > k and n ≥ 2. By usage of target-independent but population-specific reporter types, a fixed set of six optimized signalling molecules could be defined. This reporter set enables the robust generation and precise differentiation of multiple fluorescence signals in dPCRs and can be transferred to new target panels. The set which enables stable signal generation and differentiation in a specified device would allow easy transfer to new target panels.

Advanced Minimal Residual Disease Monitoring for Acute Lymphoblastic Leukemia with Multiplex Mediator Probe PCR.

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy in childhood. Minimal residual disease (MRD) monitoring is an important prognostic factor for ALL treatment response and patient stratification. MRD monitoring uses personalized real-time PCR to measure the amount of cancer cells among normal cells. Due to clonal tumor evolution or secondary rearrangement processes, MRD markers can disappear during treatment, leading to false-negative MRD results and wrong decision-making in personalized treatments. Therefore, monitoring of multiple MRD markers per patient is required. For the first time, the authors present personalized multiplex mediator probe PCR (MP PCR) for MRD monitoring in ALL. These assays can precisely quantify more MRD markers in less sample material. Therefore, clinical outcomes will be less affected by clonal tumor evolution. Personalized duplex MP PCR assays were developed for different genomic MRD markers, including immunoglobulin/T-cell receptor gene rearrangements, gene fusions, and gene deletions. One duplex assay was successfully applied in a prospective patient case and compared with hydrolysis probes. Moreover, the authors increased the multiplex level from duplex to 4-plex and still met the EuroMRD requirements for reliable quantification. In addition, the authors' MRD-MP design guidelines and multiplex workflow facilitate and accelerate MP PCR assay development. This helps the standardization of personal diagnostics.

Stringent Base Specific and Optimization-Free Multiplex Mediator Probe ddPCR for the Quantification of Point Mutations in Circulating Tumor DNA.

There is an increasing demand for optimization-free multiplex assays to rapidly establish comprehensive target panels for cancer monitoring by liquid biopsy. We present the mediator probe (MP) PCR for the quantification of the seven most frequent point mutations and corresponding wild types (KRAS and BRAF) in colorectal carcinoma. Standardized parameters for the digital assay were derived using design of experiments. Without further optimization, the limit of detection (LoD) was determined through spiking experiments with synthetic mutant DNA in human genomic DNA. The limit of blank (LoB) was measured in cfDNA plasma eluates from healthy volunteers. The 2-plex and 4-plex MP ddPCR assays showed a LoB of 0 copies/mL except for 4-plex KRAS G13D (9.82 copies/mL) and 4-plex BRAF V600E (16.29 copies/mL) and allele frequencies of 0.004% ≤ LoD ≤ 0.38% with R2 ≥ 0.98. The quantification of point mutations in patient plasma eluates (18 patients) during follow-up using the 4-plex MP ddPCR showed a comparable performance to the reference assays. The presented multiplex assays need no laborious optimization, as they use the same concentrations and cycling conditions for all targets. This facilitates assay certification, allows a fast and flexible design process, and is thus easily adaptable for individual patient monitoring.

The MRD disk: automated minimal residual disease monitoring by highly sensitive centrifugal microfluidic multiplex qPCR.

We present a proof-of-principle study on automated, highly sensitive and multiplexed qPCR quantification by centrifugal microfluidics. The MRD disk can be used for standardisation of repetitive, longitudinal assays with high requirements on reproducibility and sensitivity, such as cancer monitoring. In contrast to high-throughput qPCR automation by bulky and expensive robotic workstations we employ a small centrifugal microfluidic instrument, addressing the need of low- to mid-throughput applications. As a potential application we demonstrate automated minimum residual disease (MRD) monitoring of prognostic markers in patients with acute lymphoblastic leukaemia (ALL). The disk-workflow covers all aspects of clinical gold standard MRD quantification: generation of standard curves, specificity controls, no template controls and quantification of the ALL patient sample. We integrated a highly sensitive, colorimetric 2-plex analysis of MRD targets, as well as a 2-plex analysis of reference genes, both in parallel and in a single LabDisk cartridge. For this purpose, a systematic procedure for crosstalk- and signal-to-noise-optimisation is introduced, providing a guideline for efficient multiplex readout inside microfluidic platforms. The qPCR standard curves (n = 12/12) generated on-disk reach clinically required linearity (R2 = 98.1% to R2 = 99.8%). In three consecutive MRD disk runs with an ALL patient sample containing the two representative MRD targets VH3D3D5JH3 and VkIkde, we observe high accordance between the on-disk quantifications (48 ± 6 copies/reaction and 69 ± 6 copies/reaction) and the expected concentrations (57 copies/reaction for both targets). In comparison to the clinical gold standard of manually pipetted, singleplex assays, the MRD disk yields comparable limit of quantification (1 × 10-4) in n = 6/6 analyses (vs. n = 4/4 in gold standard) and a limit of detection (1 × 10-5) in n = 6/6 analysis (vs. n = 2/4 in gold standard). The automation reduces the risk of manual liquid handling errors, making the MRD disk an attractive solution to assure reproducibility in moderate-throughput, longitudinal gene quantification applications.

Automated serial dilutions for high-dynamic-range assays enabled by fill-level-coupled valving in centrifugal microfluidics.

We introduce a new concept for centrifugal microfluidics that enables fully automated serial dilution generation without any additional means besides temperature control. The key feature is time-independent, serial valving of mixing chambers by fill-level-coupled temperature change rate (FLC-TCR) actuated valving. The automated dilution is realized under continuous rotation which enables reliable control of wetting liquids without the need for any additional fabrication steps such as hydrophobic coating. All fluidic features are implemented in a monolithic fashion and disks are manufactured by foil thermoforming for scalable manufacturing. The new valving concept is demonstrated to reliably prevent valving if the diluted sample is not added to the mixing chamber (n = 30) and ensure valving if the dilution stage is completed (n = 15). The accuracy and precision of automated serial dilutions are verified by on-disk generation of qPCR standard curve dilutions and compared with manually generated reference dilutions. In a first step, the 5-log-stage standard curves are evaluated in a commercial qPCR thermocycler revealing a linearity of R2 ≥ 99.92% for the proposed LabDisk method vs. R2 ≥ 99.67% in manual reference dilutions. In a second step, the disk automated serial dilutions are combined with on-disk qPCR thermocycling and readout, both inside a LabDisk player. A 4-log-stage linearity of R2 ≥ 99.81% and a sensitivity of one leukemia associated ETV6-RUNX1 mutant DNA copy in a background of 100 000 wild-type DNA copies are achieved.

Promotion of osteogenic cell response using quasicovalent immobilized fibronectin on titanium surfaces: introduction of a novel biomimetic layer system.

PURPOSE: Despite the undeniable potential of cell adhesion molecules such as fibronectin to support osteogenic cell responses and consecutive dental implant healing, the most beneficial mode of application onto titanium implant surfaces still requires investigation. Unspecific fibronectin adsorption on titanium dioxide (TiO(2)) surfaces can result in low-loading, high-desorption rates and protein-metal interactions with impaired biologic activity. The aim of the present study was to monitor the osteogenic cell responses (cell adhesion, proliferation, and differentiation) specifically to fibronectin biofunctionalized TiO(2). MATERIALS AND METHODS: An innovative biomimetic streptavidin-biotin layer system allows flexible, but stable, specific binding of biotinylated biomolecules such as fibronectin on TiO(2) surfaces. Transparent glass disks were sputtered with TiO(2). The biomimetic layer system was immobilized by self-assembly and consisted of silane, biotin-derivate, streptavidin, and biotinylated fibronectin (bFN). For the control group, unbiotinylated fibronectin was directly coated onto TiO(2). Early cell adhesion dynamics were quantified using automated processing of light microscopy images within the first 24 hours. Relative mRNA expression of integrin-ß1, cyclin D1, runt-related gene 2, alkaline phosphatase, and osteocalcin was obtained using quantitative real-time polymerase chain reactions 3 and 7 days after incubation. RESULTS: Although untreated TiO(2) preserved a rather immature osteogenic phenotype, both unbiotinylated fibronectin and bFN promoted osteogenic cell adhesion and cell differentiation. In particular, runt-related gene 2 expression was significantly promoted by bFN after 3 days. In contrast, cyclin D1 expression was decreased for unbiotinylated fibronectin and bFN after 7 days. CONCLUSIONS: The introduced biomimetic layer system contributes a coherent immobilization approach of adhesion molecules with promotion of osteogenic cell response in vitro.