We report a microfluidic assay to select active severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles (VPs), which were defined as intact particles with an accessible angiotensin-converting enzyme 2 receptor binding domain (RBD) on the spike (S) protein, from clinical samples. Affinity selection of SARS-CoV-2 particles was carried out using injection molded microfluidic chips, which allow for high-scale production to accommodate large-scale screening. The microfluidic contained a surface-bound aptamer directed against the virus’s S protein RBD to affinity select SARS-CoV-2 VPs. Following selection (~94% recovery), the VPs were released from the chip’s surface using a blue light light-emitting diode (89% efficiency). Selected SARS-CoV-2 VP enumeration was carried out using reverse transcription quantitative polymerase chain reaction. The VP selection assay successfully identified healthy donors (clinical specificity = 100%) and 19 of 20 patients with coronavirus disease 2019 (COVID-19) (95% sensitivity). In 15 patients with COVID-19, the presence of active SARS-CoV-2 VPs was found. The chip can be reprogrammed for any VP or exosomes by simply changing the affinity agent.
Objective: To evaluate a microfluidics-based positive selection technology for isolating circulating trophoblasts (CTs) from peripheral blood of women whose pregnancies are affected by aneuploidy and to evaluate fetal karyotype using fluorescence in situ hybridization (FISH).Method: Ten 18-ml samples of peripheral blood were collected consecutively from pregnant women whose fetus was affected by aneuploidy. A preservation buffer was added, and the specimens were shipped overnight to the testing laboratory at ambient temperature. The specimen was infused into the fully automated microfluidics-based LiquidScan ® instrument without pre-processing. This instrument contains microfluidic chips, which are coated with antibodies (anti-huEpCAM and a proprietary antibody mixture) specific to CT surface epitopes. FISH analysis was performed on the enriched cells.Results: Fetal aneuploidy evaluated included trisomy 21 (n = 3), trisomy 18 (n = 1), trisomy 13 (n = 1), monosomy X (n = 3), and triploidy (n = 1). CTs for analysis by FISH were identified in all samples. The average number of mononucleate cells per 1 ml of whole blood was 2.11 (range 0.38-4.63) overall and was 2.67 (range 1.13-4.63) using the proprietary combination of antibodies. FISH results were concordant with the aneuploidy based on other testing in all cases. Multinucleate cells were searched for and identified in the last seven samples (average number: 0.84/1 ml). Conclusions:Our study demonstrates that the LiquidScan ® , a high-sensitivity microfluidic platform, can enrich circulating trophoblasts (mononucleate and multinucleate). FISH can then be used to detect fetal aneuploidy. Key pointsWhat is already known about this topic? � Fetal trophoblasts are found in maternal circulation during pregnancy � Existing methods used to enrich circulating trophoblasts are time-intensive and expensive
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: EVEpCAM (epithelial cell adhesion molecule, epithelial origin) and EVFAPα (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 EVEpCAM and EVFAPα, while in the case of cancer-free individuals, it yielded 0.8 and 1.25 ng of total RNA from EVEpCAM and EVFAPα, 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 EVEpCAM and EVFAPα exo-mRNA profiling using subsets of the PAM50 genes and a novel algorithm (i.e., exo-PAM50) generated 100% concordance with the tumor tissue.
Background: Despite therapeutic advances, AML remains a disease in which the majority of patients relapse after attaining remission. The presence of bone marrow MRD is associated with impending AML relapse. Prediction and prevention of relapse could improve outcomes, but most current MRD tests either require bone marrow aspirate or detect individualized molecular genetic features present in a minority of AML. Furthermore, bone marrow MRD assessments are impractical if performed frequently enough to detect most early relapses. We developed and tested a novel microfluidic chip device (MCD) that can quantitate cell numbers using automated methods and explored its ability to detect low levels of peripheral blood leukemic cells with aberrant immunophenotypes. Methods: The MCD contains sinusoidal capture channels that were coated with antibodies with specificity towards one of the commonly expressed markers found on immature myeloid cells--CD33, CD34 and CD117 (capture antigens). Initial spiking experiments used fluorescently labeled leukemia cell lines HL60 (CD33+), KG1 (CD34+), Kasumi1 (CD117+) spiked into a 5000 cell/mL suspension. Cell suspensions were passed through MCDs coated with a capture antigen known to be expressed on the tested cell line. These experiments established an efficient capture using a flow rate of 1mL/sec. Then, AML patients with an aberrant immunophenotype were enrolled in a pilot study either at the start of induction chemotherapy or prior to allogeneic stem cell transplant (SCT). For patients receiving induction chemotherapy, whole blood samples were obtained monthly starting at the time of remission assessment, or monthly starting prior to SCT. Buffered whole blood was passed through MCDs coated with a capture antigen known to be expressed on patient myeloblasts. The captured cells were then released, eluted, centrifuged and plated on a glass slide. Plated cell pellets were then labeled with fluorescent antibodies targeting surface proteins known to be aberrantly (either by lineage infidelity or asynchronous expression) expressed on the patients' AML blasts. Automated fluorescence microscopy was used to identify and quantify captured cells with the known aberrant immunophenotype of the AML blasts. Descriptive statistics described serial cell counts in patients maintaining remission and relapsing patients. Results: Of 31 patients who have been enrolled in the study to date, 13 had at least 3 post-remission MCD analyses. Of these patients, 6 had either morphologic relapse or persistent/rising marrow MRD. In these patients, there was a trend towards higher initial aberrant immunophenotype cell counts, with mean initial count = 59 (95% CI 1, 108), compared to other patients with mean initial count = 15 (95% CI 5, 25). Of 5 patients who relapsed with MCD data within 1 month prior to relapse, the mean absolute rise prior to relapse above minimum MCD cell count was 54 (95% CI 2, 105), in comparison to non-relapsing patients with mean rise of 9 (95% CI 3, 15). From the initial 16 patients, 10 underwent induction therapy (the other 6 were enrolled prior to SCT). In these 10 patients there was a non-significant association between peripheral blood aberrant immunophenotype cells and remission status following induction. A total of 8 patients underwent allogeneic SCT. Two of these patients had known bone marrow MRD at the time of SCT and had a statistically significant greater number of aberrant immunophenotypic cells pre-SCT (48 and 60) compared to the 6 MRD negative patients (median = 12, range 9, 42). Conclusions: A novel MCD assay can reliably capture and detect low numbers of AML blasts from peripheral blood using immunofluorescent imaging and automated cell counts to quantify leukemia cells with aberrant immunophenotypes. Because this method uses peripheral blood, frequent sampling is feasible and of minimal risk to patients. An ongoing clinical trial will further explore the associations between MCD-based cell enumeration and clinical endpoints in AML patients that were suggested in the pilot phase of this study. Because the MCD releases trace populations of viable cells, additional experiments, such as primary cell culturing and single cell sequencing, are possible. Figure Disclosures Foster: Bellicum Pharmaceuticals, Inc: Research Funding; Daiichi Sankyo: Consultancy; MacroGenics: Research Funding; Celgene: Research Funding. Fedoriw:Alexion Pharmaceuticals: Consultancy, Speakers Bureau. Zeidner:Celgene: Consultancy, Honoraria, Research Funding; AsystBio Laboratories: Consultancy; Merck: Research Funding; Covance: Consultancy; Pfizer: Honoraria; Agios: Honoraria; Daiichi Sankyo: Honoraria; Tolero: Honoraria, Research Funding. Coombs:Covance: Consultancy; Octopharma: Honoraria; Medscape: Honoraria; Cowen & Co.: Consultancy; Loxo: Honoraria; H3 Biomedicine: Honoraria; Dedham Group: Consultancy; Pharmacyclics: Honoraria; Abbvie: Consultancy. Mirkin:BioFluidica: Employment. Zomorrodi:BioFluidica: Employment. Toughiri:BioFluidica: Employment. Bartakova:BioFluidica: Employment. Carson:BioFluidica: Employment. Muller:BioFluidica: Employment.
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