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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.

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.