Correction: Clinical Validation of an Ultra High-Throughput Spiral Microfluidics for the Detection and Enrichment of Viable Circulating Tumor Cells
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PROVIDER: S-EPMC4193886 | biostudies-literature | 2014 Jan
REPOSITORIES: biostudies-literature
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Project description:BackgroundCirculating tumor cells (CTCs) are cancer cells that can be isolated via liquid biopsy from blood and can be phenotypically and genetically characterized to provide critical information for guiding cancer treatment. Current analysis of CTCs is hindered by the throughput, selectivity and specificity of devices or assays used in CTC detection and isolation.Methodology/principal findingsHere, we enriched and characterized putative CTCs from blood samples of patients with both advanced stage metastatic breast and lung cancers using a novel multiplexed spiral microfluidic chip. This system detected putative CTCs under high sensitivity (100%, n = 56) (Breast cancer samples: 12-1275 CTCs/ml; Lung cancer samples: 10-1535 CTCs/ml) rapidly from clinically relevant blood volumes (7.5 ml under 5 min). Blood samples were completely separated into plasma, CTCs and PBMCs components and each fraction were characterized with immunophenotyping (Pan-cytokeratin/CD45, CD44/CD24, EpCAM), fluorescence in-situ hybridization (FISH) (EML4-ALK) or targeted somatic mutation analysis. We used an ultra-sensitive mass spectrometry based system to highlight the presence of an EGFR-activating mutation in both isolated CTCs and plasma cell-free DNA (cf-DNA), and demonstrate concordance with the original tumor-biopsy samples.Conclusions/significanceWe have clinically validated our multiplexed microfluidic chip for the ultra high-throughput, low-cost and label-free enrichment of CTCs. Retrieved cells were unlabeled and viable, enabling potential propagation and real-time downstream analysis using next generation sequencing (NGS) or proteomic analysis.
| S-EPMC4085042 | biostudies-literature
Project description:Screening large numbers of target regions in multiple DNA samples for sequence variation is an important application of next-generation sequencing but an efficient method to enrich the samples in parallel has yet to be reported. We describe an advanced method that combines DNA samples using indexes or barcodes prior to target enrichment to facilitate this type of experiment. Sequencing libraries for multiple individual DNA samples, each incorporating a unique 6-bp index, are combined in equal quantities, enriched using a single in-solution target enrichment assay and sequenced in a single reaction. Sequence reads are parsed based on the index, allowing sequence analysis of individual samples. We show that the use of indexed samples does not impact on the efficiency of the enrichment reaction. For three- and nine-indexed HapMap DNA samples, the method was found to be highly accurate for SNP identification. Even with sequence coverage as low as 8x, 99% of sequence SNP calls were concordant with known genotypes. Within a single experiment, this method can sequence the exonic regions of hundreds of genes in tens of samples for sequence and structural variation using as little as 1 μg of input DNA per sample.
| S-EPMC3041504 | biostudies-literature
Project description:Here we presented a simple and effective membrane mimetic microfluidic device with antibody conjugated supported lipid bilayer (SLB) "smart coating" to capture viable circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) directly from whole blood of all stage clinical cancer patients. The non-covalently bound SLB was able to promote dynamic clustering of lipid-tethered antibodies to CTC antigens and minimized non-specific blood cells retention through its non-fouling nature. A gentle flow further flushed away loosely-bound blood cells to achieve high purity of CTCs, and a stream of air foam injected disintegrate the SLB assemblies to release intact and viable CTCs from the chip. Human blood spiked cancer cell line test showed the ~95% overall efficiency to recover both CTCs and CTMs. Live/dead assay showed that at least 86% of recovered cells maintain viability. By using 2 mL of peripheral blood, the CTCs and CTMs counts of 63 healthy and colorectal cancer donors were positively correlated with the cancer progression. In summary, a simple and effective strategy utilizing biomimetic principle was developed to retrieve viable CTCs for enumeration, molecular analysis, as well as ex vivo culture over weeks. Due to the high sensitivity and specificity, it is the first time to show the high detection rates and quantity of CTCs in non-metastatic cancer patients. This work offers the values in both early cancer detection and prognosis of CTC and provides an accurate non-invasive strategy for routine clinical investigation on CTCs.
| S-EPMC4777486 | biostudies-literature
Project description:The isolation of circulating tumor cells (CTCs) from peripheral blood with high efficiency remains a challenge hindering the utilization of CTC enrichment methods in clinical practice. Here, we propose a microfluidic channel design for the size-based hydrodynamic enrichment of CTCs from blood in an epitope-independent and high-throughput manner. The microfluidic channel comprises a spiral-shaped part followed by a widening part, incorporating successive streamlined pillars, that improves the enrichment efficiency. The design was tested against two benchmark designs, a spiral microfluidic channel and a spiral microfluidic channel followed by a widening channel without the hydrofoils, by processing 5 mL of healthy blood samples spiked with 100 MCF-7 cells. The results proved that the design with hydrofoil-shaped pillars perform significantly better in terms of recovery (recovery rate of 67.9% compared to 23.6% in spiral and 56.7% in spiral with widening section), at a cost of slightly lower white blood cell (WBC) depletion (depletion rate of 94.2% compared to 98.6% in spiral and 94.2% in spiral with widening section), at 1500 µL/min flow rate. For analytical validation, the design was further tested with A549, SKOV-3, and BT-474 cell lines, yielding recovery rates of 62.3 ± 8.4%, 71.0 ± 6.5%, and 82.9 ± 9.9%, respectively. The results are consistent with the size and deformability variation in the respective cell lines, where the increasing size and decreasing deformability affect the recovery rate in a positive manner. The analysis before and after the microfluidic chip process showed that the process does not affect cell viability.
| S-EPMC10605072 | biostudies-literature
Project description:Circulating tumor cells (CTC) isolated from the peripheral blood of cancer patients by a minimally invasive procedure provide surrogate markers of the tumor that can be repeatedly sampled. However, the selection and enumeration of CTCs by traditional methods based on surface proteins like EPCAM may not detect CTCs with a mesenchymal phenotype. Here, we employed an antibody-agnostic platform, the Parsortix® PR1 system, which enriches CTCs based on cell size and membrane deformability. We evaluated the linearity, sensitivity, and specificity of the Parsortix PR1 system in tandem with 3 downstream molecular characterization techniques using healthy donor blood spiked with cultured cell lines. Signal amplification of mRNA using a QuantiGene 25-gene assay was able to quantitate multiple epithelial genes, including CDH1, EGFR, ERBB2, KRT18, and MUC1, from high numbers of spiked cells and was able to detect KRT18 when only 50 MCF-7 or SUM190 cells were spiked into healthy donor blood. However, target amplification of mRNA by quantitative polymerase chain reaction (qPCR) showed better sensitivity; qPCR without pre-amplification was able to detect CTC-related genes in Parsortix PR1-enriched cells when as few as 5 SKBR3 cells were spiked into blood. Finally, the HTG EdgeSeq nuclease protection assay was able to profile mRNA expression of over 2,560 cancer-related genes from Parsortix PR1 enriched cells, showing enrichment in cancer signaling pathways and ERBB2, KRT19, and KRT7. Overall, the Parsortix PR1 platform may be amenable to transition into routine clinical workflows.
| S-EPMC7584183 | biostudies-literature
Project description:The analysis of circulating tumour cells (CTCs) in cancer patients could provide important information for therapeutic management. Enrichment of viable CTCs could permit performance of functional analyses on CTCs to broaden understanding of metastatic disease. However, this has not been widely accomplished. Addressing this challenge, we present a separable bilayer (SB) microfilter for viable size-based CTC capture. Unlike other single-layer CTC microfilters, the precise gap between the two layers and the architecture of pore alignment result in drastic reduction in mechanical stress on CTCs, capturing them viably. Using multiple cancer cell lines spiked in healthy donor blood, the SB microfilter demonstrated high capture efficiency (78-83%), high retention of cell viability (71-74%), high tumour cell enrichment against leukocytes (1.7-2 × 10(3)), and widespread ability to establish cultures post-capture (100% of cell lines tested). In a metastatic mouse model, SB microfilters successfully enriched viable mouse CTCs from 0.4-0.6 mL whole mouse blood samples and established in vitro cultures for further genetic and functional analysis. Our preliminary studies reflect the efficacy of the SB microfilter device to efficiently and reliably enrich viable CTCs in animal model studies, constituting an exciting technology for new insights in cancer research.
| S-EPMC4260227 | biostudies-literature
Project description:Isolation of circulating tumor cells (CTCs) from blood provides a minimally-invasive alternative for basic understanding, diagnosis, and prognosis of metastatic cancer. The roles and clinical values of CTCs are under intensive investigation, yet most studies are limited by technical challenges in the comprehensive enrichment of intact and viable CTCs with minimal white blood cell (WBC) contamination. Here, we report a novel method based on contrast of cell magnetization in biocompatible ferrofluids (a colloidal magnetic nanoparticle suspension), termed as integrated ferrohydrodynamic cell separation (iFCS), that enriches CTCs in a tumor antigen-independent and cell size variation-inclusive manner, achieves a high throughput (12 mL h-1), high recovery rate (99.08% at down to ?10 cells per mL spike ratio), and low WBC contamination (533 cells for every one milliliter blood processed) and is biocompatible. This method will enable large cohort research to define the clinical and diagnostic value of CTC subtypes.
| S-EPMC6590080 | biostudies-literature
Project description:High-throughput screening and enrichment of antibody-producing cells have many important applications. Herein, we present a droplet microfluidic approach for high-throughput screening and sorting of antibody-secreting cells using a Förster resonance electron transfer (FRET)-based assay. The FRET signal is mediated by the specific binding of the secreted antibody to two fluorescently labeled probes supplied within a droplet. Functional hybridoma cells expressing either membrane-bound or secreted monoclonal antibodies (mAbs), or both, were efficiently differentiated in less than 30 min. The antibody secretion rate by individual hybridoma cells was recorded in the range of 14,000 Abs/min, while the density of membrane-bound fraction was approximately 100 Abs/μm2. Combining the FRET assay with droplet-based single-cell sorting, an 800-fold enrichment of antigen-specific cells was achieved after one round of sorting. The presented system overcomes several key limitations observed in conventional FACS-based screening methods and should be applicable to assaying various other secreted proteins.
| S-EPMC9207670 | biostudies-literature
Project description:We present a clinical device for simple, rapid, and viable isolation of circulating tumor cells (CTCs) from cancer patient bloods. In spite of the clinical importance of CTCs, the lack of easy and non-biased isolation methods is a big hurdle for implementing CTC into clinical use. The present device made of photosensitive polymer was designed to attach to conventional syringe to isolate the CTCs at minimal resources. Its unique tapered-slits on the filter are capable not only to isolate the cell based on their size and deformability, but also to increase sample flow rate, thus achieving label-free rapid viable CTC isolation. We verified our device performance using 9 different types of cancer cells at the cell concentration from 5 to 100cells/ml, showing that the device capture 77.7% of the CTCs while maintaining their viability of 80.6%. We extended our study using the 18 blood samples from lung, colorectal, pancreatic and renal cancer patients and captured 1-172 CTCs or clustered CTCs by immunofluorescent or immunohistochemical staining. The captured CTCs were also molecularly assayed by RT-PCR with three cancer-associated genes (CK19, EpCAM, and MUC1). Those comprehensive studies proved to use our device for cancer study, thereby inaugurating further in-depth CTC-based clinical researches.
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Project description:MIPP-Seq provides an ultra-sensitive, low-cost approach for detecting and validating known and novel mutations in a highly scalable system with broad utility spanning both research and clinical diagnostic testing applications. The scalability of MIPP-Seq allows for multiplexing mutations and samples, which dramatically reduce costs of variant validation when compared to methods like ddPCR. By leveraging the power of individual analyses of multiple unique and independent reactions, MIPP-Seq can validate and precisely quantitate extremely low AAFs across multiple tissues and mutational categories including both indels and SNVs. Furthermore, using Illumina sequencing technology, MIPP-seq provides a robust method for accurate detection of novel mutations at an extremely low AAF.
2021-01-30 | GSE165780 | GEO