Enrichment of circulating trophoblasts from maternal blood using laminar microscale vortices.

OBJECTIVE: Enrichment of circulating trophoblasts (CTs) from maternal blood at week 11-13 of gestation, using laminar microscale vortices, and evaluation of the performance of the VTX-1 Liquid Biopsy System in terms of CT recovery and purity. METHOD: Eight mililiter of blood was collected from 15 pregnant women and processed with the VTX-1 Liquid Biopsy System. Y-chromosome specific quantitative PCR was performed to estimate the number of enriched male CTs. To evaluate the VTX-1 performance, the target cell recovery was characterized by spiking experiments with a trophoblast cell line. Furthermore, the total quantity of DNA after enrichment was used to calculate the number of retained maternal cells. RESULTS: Successful recovery of male CTs was established in 7 out of 10 first trimester samples from pregnant women carrying a male fetus. The number of CTs, recovered from 8 ml of blood, was estimated between two and six. Spiking experiments resulted in a CT recovery of ±35 % with ±1524 retained maternal blood cells. CONCLUSION: CTs can be enriched from maternal blood with high purity, using laminar microscale vortices, starting from 8 ml of blood.

Feasibility of single-cell analysis of model cancer and foetal cells in blood after isolation by cell picking.

The objective of the present feasibility study was to transfer single cell line cells to either microscopy slides for downstream immune characterization or to polymerase chain reaction tubes for downstream DNA quantitation. Tumour cell lines, SKBR3 and MCF7 and trophoblast cell line JEG-3 were spiked in healthy donor blood. The CytoTrack system was used to scan the spiked blood samples to identify target cells. Individual target cells were identified, picked by use of a CytoPicker and deposited to either a microscopic slide or a polymerase chain reaction tube (PCR). Single tumour cells on microscopic slides were further immunostained with human epidermal growth factor receptor 2 (Her2) and epithelial cell adhesion molecule (EpCAM). From the picked cells in polymerase chain reaction tubes, DNA was amplified, quantified and used for Short Tandem Repeat genotyping. Depositing rare cells to microscopy slides was laborious with only five cells per hour. In this study with a trained operator, the picked cells had an 80.5% recovery rate. Depositing single trophoblast cells in PCR tubes was a faster process with 10 cells in 5 min. Immunostaining of isolated cells by both Her2 and EpCAM was possible but showed varying staining intensity. Presence of trophoblasts and contaminating white blood cells in PCR tubes after cell picking was confirmed based on DNA yield and mixed Short Tandem Repeat profiles in five out of eight samples. Using the CytoPicker tool, single tumour and trophoblast cells were successfully isolated and moved from blood samples, allowing subsequent immunostaining or Short Tandem Repeat genotyping.

High-throughput single-cell screening of viable hybridomas and patient-derived antibody-secreting cells using punchable microwells.

Monoclonal antibodies (mAbs) hold significant potential as therapeutic agents and are invaluable tools in biomedical research. However, the lack of efficient high-throughput screening methods for single antibody-secreting cells (ASCs) has limited the diversity of available antibodies. Here, we introduce a novel, integrated workflow employing self-seeding microwells and an automated microscope-puncher system for the swift, high-throughput screening and isolation of single ASCs. The system allows for the individual screening and isolation of up to 6,400 cells within approximately one day, with the opportunity for parallelization and efficient upscaling. We successfully applied this workflow to both hybridomas and human patient-derived B cells, enabling subsequent clonal expansion or antibody sequence analysis through an optimized, single-cell nested reverse transcription-polymerase chain reaction (RT-PCR) procedure. By providing a time-efficient and more streamlined single ASC screening and isolation process, our workflow holds promise for driving forward progress in mAb development.

Enrichment of circulating trophoblasts from maternal blood using filtration-based Metacell® technology.

In a cell-based non-invasive prenatal test (cbNIPT), intact circulating trophoblasts (CTs) are isolated from maternal blood for subsequent genetic analysis. Enrichment of these CTs from maternal blood is the most challenging step in the cbNIPT workflow. This study aims to assess the suitability of the filtration-based Metacell® technology to enrich CTs from maternal blood at week 10 to 13 of gestation. The Metacell® technology is a novel size-based enrichment technology that combines blood filtration through 8 µm pores with an in vitro culture method. Three protocols were evaluated. First, 8 mL or 16 mL of maternal blood was filtered and subsequently cultured in vitro on the separation membrane for 3 days in RPMI 1640. In addition, 16 mL of maternal blood was filtered, and immediately processed without further culturing. Y-chromosome-specific qPCR or STR analysis was performed to evaluate the enrichment of CTs. A total of 44 samples from pregnant women, out of which 26 were carrying a male fetus, were processed. Although five enriched male fetus samples show detectable male DNA quantities, it cannot be excluded that the obtained positive signal is caused by cell-free fetal DNA sticking to the Metacell® separation membrane. In conclusion, the Metacell® technology, tested as described, is not suitable for consistent enrichment of CTs.

Kinship analysis on single cells after whole genome amplification.

Short Tandem Repeat (STR-) and Single Nucleotide Polymorphism (SNP-) genotyping have been extensively studied within forensic kinship analysis. Nevertheless, no results have been reported on kinship analysis after whole genome amplification (WGA) of single cells. This WGA step is a necessary procedure in several applications, such as cell-based non-invasive prenatal testing (cbNIPT) and pre-implantation genetic diagnosis (PGD). In cbNIPT, all putative fetal cells must be discriminated from maternal cells after enrichment from whole blood. This study investigates the efficacy and evidential value of STR- and SNP-genotyping methods for the discrimination of 24 single cells after WGA, within three families. Formaldehyde-fixed and unfixed cells are assessed in offspring-parent duos and offspring-mother-father trios. Results demonstrate that both genotyping methods can be used in all tested conditions and scenarios with 100% sensitivity and 100% specificity, with a similar evidential value for fixed and unfixed cells. Moreover, sequence-based SNP-genotyping results in a higher evidential value than length-based STR-genotyping after WGA, which is not observed using high-quality offspring bulk DNA samples. Finally, it is also demonstrated that the availability of the DNA genotypes of both parents strongly increases the evidential value of the results.

Forensic tri-allelic SNP genotyping using nanopore sequencing.

The potential and current state-of-the-art of forensic SNP genotyping using nanopore sequencing was investigated with a panel of 16 tri-allelic single nucleotide polymorphisms (SNPs), multiplexing five samples per sequencing run. The sample set consisted of three single-source human genomic reference control DNA samples and two GEDNAP samples, simulating casework samples. The primers for the multiplex SNP-loci PCR were taken from a study which researched their value in a forensic setting using conventional single-base extension technology. Workflows for multiplexed Oxford Nanopore Technologies' 1D and 1D2 sequencing were developed that provide correct genotyping of most SNP loci. Loci that are problematic for nanopore sequencing were characterized. When such loci are avoided, nanopore sequencing of forensic tri-allelic SNPs is technically feasible.

Silicon µPCR Chip for Forensic STR Profiling with Hybeacon Probe Melting Curves.

The demand to perform forensic DNA profiling outside of centralized laboratories and on the crime scene is increasing. Several criminal investigations would benefit tremendously from having DNA based information available in the first hours rather than days or weeks. However, due to the complexity and time-consuming nature of standard DNA fingerprinting methods, rapid and automated analyses are hard to achieve. We here demonstrate the implementation of an alternative DNA fingerprinting method in a single microchip. By combining PCR amplification and HyBeacon melting assays in a silicon Lab-on-a-chip (LoC), a significant step towards rapid on-site DNA fingerprinting is taken. The small form factor of a LoC reduces reagent consumption and increases portability. Additional miniaturization is achieved through an integrated heating element covering 24 parallel micro-reactors with a reaction volume of 0.14 µl each. The high level of parallelization allows the simultaneous analysis of 4 short tandem repeat (STR) loci and the amelogenin gender marker commonly included in forensic DNA analysis. A reference and crime scene sample can be analyzed simultaneously for direct comparison. Importantly, by using industry-standard semiconductor manufacturing processes, mass manufacturability can be guaranteed. Following assay design and optimization, complete 5-loci profiles could be robustly generated on-chip that are on par with those obtained using conventional benchtop real-time PCR thermal cyclers. Together, our results are an important step towards the development of commercial, mass-produced, portable devices for on-site testing in forensic DNA analysis.

Short Tandem Repeat analysis after Whole Genome Amplification of single B-lymphoblastoid cells.

To allow multiple genetic analyses on a single cell, whole genome amplification (WGA) is required. Unfortunately, studies comparing different WGA methods for downstream human identification Short Tandem Repeat (STR) analysis remain absent. Therefore, the aim of this work was to assess the performance of four commercially available WGA kits for downstream human identification STR profiling on a B-lymphoblastoid cell line. The performance was assessed using an input of one or three micromanipulated cells. REPLI-g showed a very low dropout rate, as it was the only WGA method in this study that could provide a complete STR profile in some of its samples. Although Ampli1, DOPlify and PicoPLEX did not detect all selected STR markers, they seem suitable for genetic identification in single-cell applications.

Multiplex STR amplification sensitivity in a silicon microchip.

The demand for solutions to perform forensic DNA profiling outside of centralized laboratories is increasing. We here demonstrate highly sensitive STR amplification using a silicon micro-PCR (µPCR) chip. Exploiting industry-standard semiconductor manufacturing processes, a device was fabricated that features a small form factor thanks to an integrated heating element covering three parallel micro-reactors with a reaction volume of 0.5 µl each. Diluted reference DNA samples (1 ng-31 pg) were amplified on the µPCR chip using the forensically validated AmpFISTR Identifier Plus kit, followed by conventional capillary electrophoresis. Complete STR profiles were generated with input DNA quantities down to 62 pg. Occasional allelic dropouts were observed from 31 pg downward. On-chip STR profiles were compared with those of identical samples amplified using a conventional thermal cycler for direct comparison of amplification sensitivity in a forensic setting. The observed sensitivity was in line with kit specifications for both µPCR and conventional PCR. Finally, a rapid amplification protocol was developed. Complete STR profiles could be generated in less than 17 minutes from as little as 125 pg template DNA. Together, our results are an important step towards the development of commercial, mass-produced, relatively cheap, handheld devices for on-site testing in forensic DNA analysis.

Performance of four modern whole genome amplification methods for copy number variant detection in single cells.

Whole genome amplification (WGA) has become an invaluable tool to perform copy number variation (CNV) detection in single, or a limited number of cells. Unfortunately, current WGA methods introduce representation bias that limits the detection of small CNVs. New WGA methods have been introduced that might have the potential to reduce this bias. We compared the performance of PicoPLEX DNA-Seq (Picoseq), DOPlify, REPLI-g and Ampli-1 WGA for aneuploidy screening and copy number analysis using shallow whole genome massively parallel sequencing (MPS), starting from single or a limited number of cells. Although the four WGA methods perform differently, they are all suited for this application.

STR profiling and Copy Number Variation analysis on single, preserved cells using current Whole Genome Amplification methods.

The growing interest in liquid biopsies for cancer research and cell-based non-invasive prenatal testing (NIPT) invigorates the need for improved single cell analysis. In these applications, target cells are extremely rare and fragile in peripheral circulation, which makes the genetic analysis very challenging. To overcome these challenges, cell stabilization and unbiased whole genome amplification are required. This study investigates the performance of four WGA methods on single or a limited number of cells after 24 hour of Streck Cell-Free DNA BCT preservation. The suitability of the DNA, amplified with Ampli1, DOPlify, PicoPLEX and REPLI-g, was assessed for both short tandem repeat (STR) profiling and copy number variant (CNV) analysis after shallow whole genome massively parallel sequencing (MPS). Results demonstrate that Ampli1, DOPlify and PicoPLEX perform well for both applications, with some differences between the methods. Samples amplified with REPLI-g did not result in suitable STR or CNV profiles, indicating that this WGA method is not able to generate high quality DNA after Streck Cell-Free DNA BCT stabilization of the cells.