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Extracellular vesicles (EVs) carry RNA cargo that is believed to be associated with the cell-of-origin and thus have the potential to serve as a minimally invasive liquid biopsy marker for supplying molecular information to guide treatment decisions (i.e., precision medicine). We report the affinity isolation of EV subpopulations with monoclonal antibodies attached to the surface of a microfluidic chip that is made from a plastic to allow for high-scale production. The EV microfluidic affinity purification (EV-MAP) chip was used for the isolation of EVs sourced from two-orthogonal cell types and was demonstrated for its utility in a proof-of-concept application to provide molecular subtyping information for breast cancer patients. The orthogonal selection process better recapitulated the epithelial tumor microenvironment by isolating two subpopulations of EVs: EV(EpCAM) (epithelial cell adhesion molecule, epithelial origin) and EV(FAPα) (fibroblast activation protein α, mesenchymal origin). The EV-MAP provided recovery >80% with a specificity of 99 ± 1% based on exosomal mRNA (exo-mRNA) and real time–droplet digital polymerase chain reaction results. When selected from the plasma of healthy donors and breast cancer patients, EVs did not differ in size or total RNA mass for both markers. On average, 0.5 mL of plasma from breast cancer patients yielded ~2.25 ng of total RNA for both EV(EpCAM) and EV(FAPα), while in the case of cancer-free individuals, it yielded 0.8 and 1.25 ng of total RNA from EV(EpCAM) and EV(FAPα), respectively. To assess the potential of these two EV subpopulations to provide molecular information for prognostication, we performed the PAM50 test (Prosigna) on exo-mRNA harvested from each EV subpopulation. Results suggested that EV(EpCAM) and EV(EAPα) exo-mRNA profiling using subsets of the PAM50 genes and a novel algorithm (i.e., exo-PAM50) generated 100% concordance with the tumor tissue.

Currently there is no in vitro diagnostic test for acute ischemic stroke (AIS), yet rapid diagnosis is crucial for effective thrombolytic treatment. We previously demonstrated the utility of CD8(+) T-cells’ mRNA expression for AIS detection; however extracellular vesicles (EVs) were not evaluated as a source of mRNA for AIS testing. We now report a microfluidic device for the rapid and efficient affinity-enrichment of CD8(+) EVs and subsequent EV’s mRNA analysis using droplet digital PCR (ddPCR). The microfluidic device contains a dense array of micropillars modified with anti-CD8α monoclonal antibodies that enriched 158 ± 10 nm sized EVs at 4.3 ± 2.1 × 109 particles/100 µL of plasma. Analysis of mRNA from CD8(+) EVs and their parental T-cells revealed correlation in the expression for AIS-specific genes in both cell lines and healthy donors. In a blinded study, 80% test positivity for AIS patients and controls was revealed with a total analysis time of 3.7 h., Wijerathne et al develop an approach to quantify mRNA amounts in extracellular vesicles (EVs) from plasma using a microfluidics device to enrich EVs. They show this method allows for the correlation of specific mRNAs with acute ischemic stroke pointing towards potential clinical use.

Circulating tumor cells consist of phenotypically distinct subpopulations that originate from the tumor microenvironment. We report a circulating tumor cell dual selection assay that uses discrete microfluidics to select circulating tumor cell subpopulations from a single blood sample; circulating tumor cells expressing the established marker epithelial cell adhesion molecule and a new marker, fibroblast activation protein alpha, were evaluated. Both circulating tumor cell subpopulations were detected in metastatic ovarian, colorectal, prostate, breast, and pancreatic cancer patients and 90% of the isolated circulating tumor cells did not co-express both antigens. Clinical sensitivities of 100% showed substantial improvement compared to epithelial cell adhesion molecule selection alone. Owing to high purity (>80%) of the selected circulating tumor cells, molecular analysis of both circulating tumor cell subpopulations was carried out in bulk, including next generation sequencing, mutation analysis, and gene expression. Results suggested fibroblast activation protein alpha and epithelial cell adhesion molecule circulating tumor cells are distinct subpopulations and the use of these in concert can provide information needed to navigate through cancer disease management challenges., Diagnostics: Dual selection improves circulating tumor cell assay Using two selection markers, instead of one, can improve the sensitivity of a blood test for circulating tumor cells (CTCs). Steven A. Soper from the University of Kansas in Lawrence, USA, and colleagues analyzed blood samples from patients with cancers of the pancreas, colon, breast, ovaries and prostate, as well blood from healthy donors and those with benign disease. They ran each patient’s blood through two microfluidic devices, one chip targeting the established marker epithelial cell adhesion molecule (EpCAM) and another targeting a new marker, fibroblast activation protein alpha (FAPα). Doing so detected CTCs in nearly all the cancer patients—a substantial improvement over testing for EpCAM CTCs alone. Molecular and genetic analyses of the CTCs showed that those expressing EpCAM and FAPα are distinct subpopulations, and testing for both could prove valuable for clinical management.

We present a critical review of microfluidic technologies and material effects on the analyses of circulating tumour cells (CTCs) selected from the peripheral blood of cancer patients. CTCs are a minimally invasive source of clinical information that can be used to prognose patient outcome, monitor minimal residual disease, assess tumour resistance to therapeutic agents, and potentially screen individuals for the early diagnosis of cancer. The performance of CTC isolation technologies depends on microfluidic architectures, the underyling principles of isolation, and the choice of materials. In this review, we present a critical review of the fundamental principles used in these technologies and discuss their performance. We also give context to how CTC isolation technologies enable downstream analysis of selected CTCs in terms of detecting genetic mutations and gene expression that could be used to gain information that may affect patient outcome.

In the context of oncology, liquid biopsies consist of harvesting cancer biomarkers, such as circulating tumor cells (CTCs), tumor-derived cell-free DNA (ctDNA), and extracellular vesicles (EVs), from bodily fluids. These biomarkers provide a source of clinically-actionable molecular information that can enable precision medicine. Herein, we review technologies for the molecular profiling of liquid biopsy markers with special emphasis on the analysis of low abundant markers from mixed-populations.

We report a novel strategy to enzymatically release affinity-selected cells, such as circulating tumor cells (CTCs), from surfaces with high efficiency (~90%) while maintaining cell viability (>85%). The strategy utilizes single-stranded DNAs that link a capture antibody to the surfaces of a CTC selection device. The DNA linkers contain a uracil residue that can be cleaved.

Parallel channel configurations, such as Z-type, used to distribute reagents in planar fuel cells provide lower overall pressure drop as compared to other channel designs. However, due to their inherent characteristics, flow maldistribution in parallel configurations is commonly observed and leads to starvation of reagents in middle channels. In addition, the Reynolds number dependent minor losses at branching tee junctions may cause asymmetric flow non-uniformity and reagent imbalance between the cathode and anode. Herein, we present a universal and simple optimization method to simultaneously reduce flow maldistribution, asymmetry, and parasitic pressure in Z-type parallel configurations of fuel cells or fuel cell stacks that has improved scalability relative to previous methods. A discrete model's governing equations were reduced to yield geometric ratios between headers. Increasing header widths to satisfy these ratios reduced flow maldistribution without modifying parallel channel geometry as validated by computation fluid dynamics (CFD) simulations. Furthermore, decreased Reynolds numbers throughout the headers reduced minor pressure drops and flow distribution asymmetry. We offer several methods to reduce the optimized geometry's footprint, including an adaptation of the discontinuous design.

The need to activate thermoplastic surfaces using robust and efficient methods has been driven by the fact that replication techniques can be used to produce microfluidic devices in a high production mode and at low cost, making polymer microfluidics invaluable for in vitro diagnostics, such as circulating tumor cell (CTC) analysis, where device disposability is critical to mitigate artifacts associated with sample carryover. Modifying the surface chemistry of thermoplastic devices through activation techniques can be used to increase the wettability of the surface or to produce functional scaffolds to allow for the covalent attachment of biologics, such as antibodies for CTC recognition. Extensive surface characterization tools were used to investigate UV activation of various surfaces to produce uniform and high surface coverage of functional groups, such as carboxylic acids in microchannels of different aspect ratios. We found that the efficiency of the UV activation process is highly dependent on the microchannel aspect ratio and the identity of the thermoplastic substrate. Colorimetric assays and fluorescence imaging of UV-activated microchannels following EDC/NHS coupling of Cy3-labeled oligonucleotides indicated that UV-activation of a PMMA microchannel with an aspect ratio of ∼3 was significantly less efficient toward the bottom of the channel compared to the upper sections. This effect was a consequence of the bulk polymer's damping of the modifying UV radiation due to absorption artifacts. In contrast, this effect was less pronounced for COC. Moreover, we observed that after thermal fusion bonding of the device's cover plate to the substrate, many of the generated functional groups buried into the bulk rendering them inaccessible. The propensity of this surface reorganization was found to be higher for PMMA compared to COC. As an example of the effects of material and microchannel aspect ratios on device functionality, thermoplastic devices for the selection of CTCs from whole blood were evaluated, which required the immobilization of monoclonal antibodies to channel walls. From our results, we concluded the CTC yield and purity of isolated CTCs were dependent on the substrate material with COC producing the highest clinical yields for CTCs as well as better purities compared to PMMA.

We present a novel microfluidic solid-phase extraction (μSPE) device for the affinity enrichment of biotinylated membrane proteins from whole cell lysates. The device offers features that address challenges currently associated with the extraction and purification of membrane proteins from whole cell lysates, including the ability to release the enriched membrane protein fraction from the extraction surface so that they are available for downstream processing. The extraction bed was fabricated in PMMA using hot embossing and was comprised of 3,600 micropillars. Activation of the PMMA micropillars by UV/O3 treatment permitted generation of surface-confined carboxylic acid groups and the covalent attachment of NeutrAvidin onto the μSPE device surfaces, which was used to affinity select biotinylated MCF-7 membrane proteins directly from whole cell lysates. The inclusion of a disulfide linker within the biotin moiety permitted release of the isolated membrane proteins via DTT incubation. Very low levels (~20 fmol) of membrane proteins could be isolated and recovered with ~89% efficiency with a bed capacity of 1.7 pmol. Western blotting indicated no traces of cytosolic proteins in the membrane protein fraction as compared to significant contamination using a commercial detergent-based method. We highlight future avenues for enhanced extraction efficiency and increased dynamic range of the μSPE device using computational simulations of different micropillar geometries to guide future device designs.

We present a novel approach for characterizing surfaces utilizing super-resolution fluorescence microscopy with subdiffraction limit spatial resolution. Thermoplastic surfaces were activated by UV/O3 or O2 plasma treatment under various conditions to generate pendant surface-confined carboxylic acids (–COOH). These surface functional groups were then labeled with a photoswitchable dye and interrogated using single-molecule, localization-based, super-resolution fluorescence microscopy to elucidate the surface heterogeneity of these functional groups across the activated surface. Data indicated nonuniform distributions of these functional groups for both COC and PMMA thermoplastics with the degree of heterogeneity being dose dependent. In addition, COC demonstrated relative higher surface density of functional groups compared to PMMA for both UV/O3 and O2 plasma treatment. The spatial distribution of –COOH groups secured from super-resolution imaging were used to simulate nonuniform patterns of electroosmotic flow in thermoplastic nanochannels. Simulations were compared to single-particle tracking of fluorescent nanoparticles within thermoplastic nanoslits to demonstrate the effects of surface functional group heterogeneity on the electrokinetic transport process.

We offer a simple way to include energy dissipation in small spatial–dimensional quantum dynamics simulations of a primary quantum system coupled to its surroundings. We show how a quantum Langevin equation can be generated by including a friction operator, F ˆ , in the primary quantum system’s Hamiltonian operator. We demonstrate this approach’s computational ease and flexibility through numerical results on both harmonic and anharmonic primary quantum systems. Also, we show how the present approach can be easily generalized to non-Markovian, ‘memory’ friction. Thus, the approach presented in this work can be used to generate a quantum generalized Langevin equation.

Microsystem-based technologies are providing new opportunities in the area of in vitro diagnostics due to their ability to provide process automation enabling point-of-care operation. As an example, microsystems used for the isolation and analysis of circulating tumor cells (CTCs) from complex, heterogeneous samples in an automated fashion with improved recoveries and selectivity are providing new opportunities for this important biomarker. Unfortunately, many of the existing microfluidic systems lack the throughput capabilities and/or are too expensive to manufacture to warrant their widespread use in clinical testing scenarios. Here, we describe a disposable, all-polymer, microfluidic system for the high-throughput (HT) isolation of CTCs directly from whole blood inputs. The device employs an array of high aspect ratio (HAR), parallel, sinusoidal microchannels (25 µm × 150 µm; W × D; AR = 6.0) with walls covalently decorated with anti-EpCAM antibodies to provide affinity-based isolation of CTCs. Channel width, which is similar to an average CTC diameter (12–25 µm), plays a critical role in maximizing the probability of cell/wall interactions and allows for achieving high CTC recovery. The extended channel depth allows for increased throughput at the optimized flow velocity (2 mm/s in a microchannel); maximizes cell recovery, and prevents clogging of the microfluidic channels during blood processing. Fluidic addressing of the microchannel array with a minimal device footprint is provided by large cross-sectional area feed and exit channels poised orthogonal to the network of the sinusoidal capillary channels (so-called Z-geometry). Computational modeling was used to confirm uniform addressing of the channels in the isolation bed. Devices with various numbers of parallel microchannels ranging from 50 to 320 have been successfully constructed. Cyclic olefin copolymer (COC) was chosen as the substrate material due to its superior properties during UV-activation of the HAR microchannels surfaces prior to antibody attachment. Operation of the HT-CTC device has been validated by isolation of CTCs directly from blood secured from patients with metastatic prostate cancer. High CTC sample purities (low number of contaminating white blood cells, WBCs) allowed for direct lysis and molecular profiling of isolated CTCs.

We report the design and performance of a polymer microfluidic device that can affinity select multiple types of biological cells simultaneously with sufficient recovery and purity to allow for the expression profiling of mRNA isolated from these cells. The microfluidic device consisted of four independent selection beds with curvilinear channels that were 25 μm wide and 80 μm deep and were modified with antibodies targeting antigens specifically expressed by two different cell types. Bifurcated and Z-configured device geometries were evaluated for cell selection. As an example of the performance of these devices, CD4+ T-cells and neutrophils were selected from whole blood as these cells are known to express genes found in stroke-related expression profiles that can be used for the diagnosis of this disease. CD4+ T-cells and neutrophils were simultaneously isolated with purities >90% using affinity-based capture in cyclic olefin copolymer (COC) devices with a processing time of ∼3 min. In addition, sufficient quantities of the cells could be recovered from a 50 μL whole blood input to allow for reverse transcription-polymerase chain reaction (RT-PCR) following cell lysis. The expression of genes from isolated T-cells and neutrophils, such as S100A9, TCRB, and FPR1, was evaluated using RT-PCR. The modification and isolation procedures demonstrated here can also be used to analyze other cell types as well where multiple subsets must be interrogated.

In this manuscript, we discuss the development and clinical use of a thermoplastic modular microsystem for the high-throughput analysis of CTCs directly from whole blood. The modular system offers some innovative features that address challenges currently associated with many CTC platforms; it can exhaustively process 7.5 ml of blood in less than 45 min with recoveries >90%. In addition, the system automates the post-selection CTC processing steps and thus, significantly reduces assay turnaround time (from selection to enumeration 8 h for many reported CTC platforms). The system is comprised of 3 functional modules including; (i) a thermoplastic CTC selection module composed of high aspect ratio (30 μm × 150 μm) channels containing anti-EpCAM antibodies that is scalable in terms of throughput by employing channel numbers ranging from 50 to 320 – the channel number is user selected to accommodate the volume of blood that must be processed; (ii) an impedance sensor module for label-less CTC counting; and (iii) a staining and imaging module for the placement of released cells into a 2D array within a common imaging plane for phenotypic identification. To demonstrate the utility of this system, blood samples from patients with local resectable and metastatic pancreatic ductal adenocarcinoma (PDAC) were analyzed. We demonstrate the ability to select EpCAM positive CTCs from PDAC patients in high purity (>86%) and with excellent yields (mean = 53 CTCs per ml for metastatic PDAC patients) using our modular system. In addition, we demonstrate the ability to detect CTCs in PDAC patients with local resectable disease (mean = 11 CTCs per ml).

Background: The detection of minimal residual disease (MRD) following therapy in acute myeloid leukemia (AML) is associated with increased risk of relapse and poor survival and can influence clinical decisions regarding treatment. Multi-parameter flow cytometry can assess MRD in ~90% of AML cases (due to aberrant surface antigen expression); however, its poor sensitivity typically requires bone marrow aspirates, which are invasive and cannot be obtained frequently. Polymerase chain reaction (PCR) assays are highly sensitive in detecting AML-associated mutations, but significant sample processing and assay development are needed to detect patient-specific mutations, which curbs the broad applicability of PCR for the majority of AML patients. Here we present a microfluidic chip assay (Figure 1) with high sensitivity for detecting low levels of aberrant antigen expressing blasts in peripheral blood, thereby enabling frequent MRD monitoring for high-risk AML patients undergoing allogeneic stem cell transplant (SCT). Early detection of MRD in AML post-SCT can influence clinical decisions, such as adjustments in immunosuppression or administration of donor lymphocytes. Methods: Subjects had 3 mL of peripheral blood drawn into an EDTA tube every 2 weeks following their SCT from day 14 through day 98 and monthly through day 365 post-SCT. The blood was injected into microfluidic chips (Figure 1), each containing 50 flow channelsthat were chemically modified with capture antibodies specific for human CD34, CD33 or CD117 - surface antigens commonly expressed by AML blasts. Following cell selection, fluorescently-labeled antibodies specific for 1) the capture antibody, 2) an aberrant lineage marker (e.g. CD7, CD56) that was known to be present on the patients AML blasts and 3) CD45, were injected into the chips. Cells were then released for off-chip fluorescence microscopy and enumerated with the results compared to conventional MRD assays, ongoing treatments and clinical outcomes. Results: 7 patients were enrolled with 4 having reportable data at the time of submission. Pt#1 had AML with NPM1 and FLT3-ITD mutations. NPM1 PCR classified Pt#1 as MRD(-) 51 days after SCT, while the microfluidic assay was MRD(+) at day 48 with 81 aberrant cells/mL. The aberrant cell count began to steadily increase at day 77 that persisted until Pt#1 was diagnosed with hematologic relapse on day 85 and died at day 95 (Figure 2). Pt#2 is currently 216 days post-SCT and has had persistent low levels (5 - 57 cells/mL) of aberrant cells since day 64. This patient recently developed GVHD and is being treated with corticosteroids and has no evidence of hematologic relapse at this time. Pt#3 developed high level aberrant cells (1066 cells/mL) at day 35 post SCT. These values dropped while the patient was treated with valganciclovirfor CMV viremia (22 cells/mL at day 47), but the high level of AML blasts returned upon completion of therapy (443 cells/mL at day 105). This patient developed multi-organ system failure and at the time of death (day 124), circulating immature cells were detectable in the peripheral blood. Pt#4 has AML with an NPM1 type A mutation. The patient was MRD(+) as determined by NPM1 PCR analysis (checked 3 times) and by the microfluidic assay (checked 7 times); however, on the most recent assessment at day 125, both assays were MRD(-). Conclusion: A microfluidic assay was able to isolate trace quantities of circulating AML blasts ( Figure 1. (A-B) Microfluidic chips and (C-D) assay workflow for frequent MRD analysis in AML. A single blood sample was split 3-ways so that each chip processed the same volume. Figure 1. (A-B) Microfluidic chips and (C-D) assay workflow for frequent MRD analysis in AML. A single blood sample was split 3-ways so that each chip processed the same volume. Figure 2. Disease progression for Pt#1 determined by (A-C) the microfluidic assay and (D-E) conventional diagnostics. Figure 2. Disease progression for Pt#1 determined by (A-C) the microfluidic assay and (D-E) conventional diagnostics. Disclosures Soper: Biofluidica: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.