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Nirmatrelvir is a potent and selective severe acute respiratory syndrome‐coronavirus 2 (SARS‐CoV‐2) main protease inhibitor. Nirmatrelvir co‐packaged with ritonavir (as PAXLOVID) received US Food and Drug Administration (FDA) Emergency Use Authorization (EUA) on December 22, 2021, as an oral treatment for coronavirus disease 2019 (COVID‐19) and subsequent new drug application approval on May 25, 2023. Pharmacokinetic (PK) capillary blood sampling at‐home using Tasso‐M20 micro‐volumetric sampling device was implemented in the program, including three phase II/III outpatient and several clinical pharmacology studies supporting the EUA. The at‐home sampling complemented venous blood sampling procedures to enrich the PK dataset, to decrease the need for patients' site visit for PK sampling, and to allow different sampling approaches for flexibility and convenience. To demonstrate concordance/equivalence, bridging between venous plasma and Tasso dried blood results was conducted by comparing concentrations and derived PK parameters from both sampling approaches. In addition, a two‐compartment population PK model was utilized to bridge the plasma and Tasso data by estimating the PK parameters using blood‐to‐plasma ratio as a slope parameter. Operational challenges were successfully managed to implement at‐home PK sampling in global phase II/III trials. Sample quality was generally very good with less than 3% samples deemed as "not usable" from over 800 samples collected in all the studies. Experience gained from sites and patients will guide future broader implementations. [ABSTRACT FROM AUTHOR]

Trigger valves are fundamental features in capillary-driven microfluidic systems that stop fluid at an abrupt geometric expansion and release fluid when there is flow in an orthogonal channel connected to the valve. The concept was originally demonstrated in closed-channel capillary circuits. We show here that trigger valves can be successfully implemented in open channels. We also show that a series of open-channel trigger valves can be placed alongside or opposite a main channel resulting in a layered capillary flow. We developed a closed form model for the dynamics of the flow at trigger valves based on the concept of average friction length and successfully validated the model against experiments. For the main channel, we discuss layered flow behavior in the light of the Taylor-Aris dispersion theory and in the channel turns by considering Dean theory of mixing. This work has potential applications in autonomous microfluidics systems for biosensing, at-home or point-of-care sample preparation devices, hydrogel patterning for 3D cell culture and organ-on-a-chip models., Competing Interests: A.B.T. reports filing multiple patents through the University of Washington and A.B.T. received a gift to support research outside the submitted work from Ionis Pharmaceuticals. E.B. is an inventor on multiple patents filed by Tasso, Inc., the University of Washington, and the University of Wisconsin-Madison. T.M.N. has ownership in Tasso, Inc.; E.B. has ownership in Tasso, Inc., Salus Discovery, LLC, and Seabright, LLC and is employed by Tasso, Inc.; and A.B.T. has ownership in Seabright, LLC; however, this research is not related to these companies. The terms of this arrangement have been reviewed and approved by the University of Washington in accordance with its policies governing outside work and financial conflicts of interest in research. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Sperm cryopreservation is important for many individuals across the globe. Recent studies show that vitrification is a valuable approach for maintaining sperm quality after freeze-thawing processes and requires sub-microliter to microliter volumes. A major challenge for the adoption of vitrification in fertility laboratories is the ability to pipette small volumes of sample. Here, we present an open droplet generator that leverages open-channel microfluidics to passively generate sub-microliter to microliter volumes of purified human sperm samples and preserves sperm kinematics. We conclude that our platform is compatible with human sperm, an important foundation for future implementation of vitrification in fertility laboratories., Competing Interests: T.M.N., J.W.K., U.N.L., E.B., and A.B.T. filed patent Open Microfluidic Channel Design for Passive Droplet Formation and Manipulation May 23, 2022 through the University of Washington. A.B.T. and U.N.L. report filing multiple patents through the University of Washington and A.B.T. received a gift to support research outside the submitted work from Ionis Pharmaceuticals. E.B. is an inventor on multiple patents filed by Tasso, Inc., the University of Washington, and the University of Wisconsin-Madison. T.M.N. has ownership in Tasso, Inc.; E.B. has ownership in Tasso, Inc., Salus Discovery, LLC, and Seabright, LLC and is employed by Tasso, Inc.; and A.B.T. has ownership in Seabright, LLC; however, this research is not related to these companies. The terms of this arrangement have been reviewed and approved by the University of Washington in accordance with its policies governing outside work and financial conflicts of interest in research. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Extracellular Vesicles (EVs) are membrane-derived vesicles shed by cells into the extracellular space that play key roles in intercellular communication and other biological processes. As membrane-bound cargos of nucleic acids and other proteins that are abundantly found in virtually every biofluid including blood, urine, and saliva, EVs are widely regarded as promising biomarkers for disease detection. While it is an increasingly promising biofluid from which to isolate EVs, saliva poses challenges due its complexity and heterogeneity-cells, debris, and other proteins can inhibit the isolation of EVs by traditional platforms. Here, we employ the CandyCollect, a lollipop-inspired sampling device with open microfluidic channels, as a non-invasive and patient-friendly alternative for the capture of salivary EVs. The CandyCollect simplifies sample preparation by effectively pre-concentrating EVs on the device surface before EVs are eluted off of the CandyCollect, labeled with cholesterol-tagged oligonucleotides, and subsequently detected by qPCR with primers specific for the tagged oligos to enumerate the relative number of EVs. We demonstrate that downstream EV cargo analysis can be performed using Simoa. Overall, the CandyCollect ushers a new method to capture, enumerate, and analyze salivary EVs.

Cell-laden hydrogel constructs suspended between pillars are powerful tools for modeling tissue structure and physiology, though current fabrication techniques often limit them to uniform compositions. In contrast, tissues are complex in nature with spatial arrangements of cell types and extracellular matrices. Thus, we present Suspended Tissue Open Microfluidic Patterning (STOMP), which utilizes a removable, open microfluidic patterning channel to pattern multiple spatial regions across a single suspended tissue. The STOMP platform contains capillary pinning features along the open channel that controls the fluid front, allowing multiple cell and extracellular matrix precursors to be pipetted into one tissue. We have used this technique to pattern suspended tissues with multiple regional components using a variety of native and synthetic extracellular matrices, including fibrin, collagen, and poly(ethylene glycol). Here, we demonstrate that STOMP models a region of fibrosis in a functional heart tissue and a bone-ligament junction in periodontal tissues. Additionally, the STOMP platform can be customized to allow patterning of suspended cores and more spatial configurations, enhancing its utility in complex tissue modeling. STOMP is a versatile technique for generating suspended tissue models with increased control over cell and hydrogel composition to model interfacial tissue regions in a suspended tissue., Competing Interests: Conflicts of interest AJH, LGB, AJG, PM, ARV, TPL, SHN, AL, JMW, RCB, EB, CAD, NJS, and ABT filed patent 18/669,367 and AJH, LGB, JMW, ARV, CAD, NJS, EEB, EB, and ABT filed patent 63/665,194 through the University of Washington on STOMP and a related technology. ABT reports filing multiple patents through the University of Washington and receiving a gift to support research from Ionis Pharmaceuticals. EB and ABT have ownership in Seabright, LLC, which will advance new tools for diagnostics and clinical research. EB has ownership in Salus Discovery, LLC, and Tasso, Inc. and is employed by Tasso, Inc. NJS has ownership in Stasys Medical Corporation, Inc. and has ownership and is a scientific advisor in Curi Bio, Inc. Technologies from Salus Discovery, LLC, Tasso, Inc., Stasys Medical Corporation, Inc, and Curi Bio, Inc. are not included in this publication. EB is an inventor on multiple patents filed by Tasso, Inc., the University of Washington, and the University of Wisconsin-Madison. The terms of this arrangement have been reviewed and approved by the University of Washington in accordance with its policies governing outside work and financial conflicts of interest in research.

The CandyCollect device is a lollipop-inspired open fluidic oral sampling device designed to provide a comfortable user sampling experience. We demonstrate that the CandyCollect device can be coupled with a rapid antigen detection test (RADT) kit designed for Group A Streptococcus (GAS). Through in vitro experiments with pooled saliva spiked with Streptococcus pyogenes we tested various reagents and elution volumes to optimize the RADT readout from CandyCollect device samples. The resulting optimized protocol uses the kit-provided reagents and lateral flow assay (LFA) while replacing the kit's pharyngeal swab with the CandyCollect device, reducing the elution solution volume, and substituting the tube used for elution to accommodate the CandyCollect device. Positive test results were detected by eye with bacterial concentrations as low as the manufacturer's "minimal detection limit" - 1.5×10 5 CFU/mL. LFA strips were also scanned and quantified with image analysis software to determine the signal-to-baseline ratio (SBR) and categorize positive test results without human bias. We tested our optimized protocol for integrating CandyCollect and RADT using CandyCollect clinical samples from pediatric patients (n=6) who were previously diagnosed with GAS pharyngitis via pharyngeal swabs tested with RADT as part of their clinical care. The LFA results of these CandyCollect devices and interspersed negative controls were determined by independent observers, with positive results obtained in four of the six participants on at least one LFA replicate. Taken together, our results show that CandyCollect devices from children with GAS pharyngitis can be tested using LFA rapid tests., Competing Interests: Conflict of Interest Disclosures Ashleigh B. Theberge, Xiaojing Su, Erwin Berthier, and Sanitta Thongpang filed patent 63/152,103 (International Publication Number: WO 2022/178291 Al) through the University of Washington on the CandyCollect oral sampling device. J. Carlos Sanchez, Timothy R. Robinson, Ayokunle O. Olanrewaju, Erwin Berthier, and Ashleigh B. Theberge filed patent 63/683,571 through the University of Washington on a related platform. Ashleigh B. Theberge reports filing multiple patents through the University of Washington and receiving a gift to support research outside the submitted work from Ionis Pharmaceuticals. Erwin Berthier is an inventor on multiple patents filed by Tasso, Inc., the University of Washington, and the University of Wisconsin. Sanitta Thongpang has ownership in Salus Discovery, LLC, and Tasso, Inc. Erwin Berthier has ownership in Salus Discovery, LLC, and Tasso, Inc. and is employed by Tasso, Inc. However, this research is not related to these companies. Sanitta Thongpang, Erwin Berthier, and Ashleigh B. Theberge have ownership in Seabright, LLC, which will advance new tools for diagnostics and clinical research, potentially including the CandyCollect device. The terms of this arrangement have been reviewed and approved by the University of Washington in accordance with its policies governing outside work and financial conflicts of interest in research. The other authors have no conflicts of interest to disclose.

State the Purpose: Obtaining high-quality samples to diagnose streptococcal pharyngitis in pediatric patients is challenging due to discomfort associated with traditional pharyngeal swabs. This may cause reluctance to go to the clinic, inaccurate diagnosis, or inappropriate treatment for children with sore throat. Here, we determined the efficacy of CandyCollect, a lollipop-inspired open-microfluidic pathogen collection device, to capture Group A Streptococcus (GAS) and compare user preference for CandyCollect, conventional pharyngeal swabs, or mouth swabs in children with pharyngitis and their caregivers., Results: All child participants (30/30) were positive for GAS by qPCR on both the mouth swab and CandyCollect. Caregivers ranked CandyCollect as a good sampling method overall (27/30), and all caregivers (30/30) would recommend CandyCollect for children 5 years and older. Twenty-three of 30 children "really like" the taste and 24/30 would prefer to use CandyCollect if a future test were needed. All caregivers (30/30) and most children (28/30) would be willing to use CandyCollect at home., Conclusion: All participants tested positive for GAS on all three collection methods (pharyngeal swab, mouth swab, and CandyCollect). While both caregivers and children like CandyCollect, some caregivers would prefer a shorter collection time. Future work includes additional studies with larger cohorts presenting with pharyngitis of unknown etiology and shortening collection time while maintaining the attractive form of the device., Translational Impact Statement: Obtaining oral samples for the diagnosis of streptococcal pharyngitis is of great importance for children. To address the challenges associated with traditional pharyngeal swab sampling, we developed the CandyCollect device, a lollipop-inspired open mesofluidic saliva sampling system. In this study, saliva samples were collected from children, aged 5-14 years, with CandyCollect and mouth swabs and analyzed via qPCR. The results show CandyCollect is the child preferred collection tool and had 100% concordance with the results from traditional diagnosis methods as part of their clinical care.

Background: Early host immunity to acute respiratory infections (ARIs) is heterogenous, dynamic, and critical to an individual's infection outcome. Due to limitations in sampling frequency/timepoints, kinetics of early immune dynamics in natural human infections remain poorly understood. In this nationwide prospective cohort study, we leveraged a Tasso-SST based self-blood collection and stabilization tool ( home RNA) to profile detailed kinetics of the presymptomatic to convalescence host immunity to contemporaneous respiratory pathogens., Methods: We enrolled non-symptomatic adults with recent exposure to ARIs who subsequently tested negative (exposed-uninfected) or positive for respiratory pathogens. Participants self-collected blood and nasal swabs daily for seven consecutive days followed by weekly blood collection for up to seven additional weeks. Symptom burden was assessed during each collection. Nasal swabs were tested for SARS-CoV-2 and common respiratory pathogens. 92 longitudinal blood samples spanning the presymptomatic to convalescence phase of eight SARS-CoV-2-infected participants and 40 interval-matched samples from four exposed-uninfected participants were subjected to high-frequency longitudinal profiling of 785 immune genes. Generalized additive mixed models (GAMM) were used to identify temporally dynamic genes from the longitudinal samples and linear mixed models (LMM) were used to identify baseline differences between exposed-infected (n = 8), exposed-uninfected (n = 4), and uninfected (n = 13) participant groups., Findings: Between June 2021 - April 2022, 68 participants across 26 U.S. states completed the study and self-collected a total of 691 and 466 longitudinal blood and nasal swab samples along with 688 symptom surveys. SARS-CoV-2 was detected in 17 out of 22 individuals with study-confirmed respiratory infection, of which five were still presymptomatic or pre-shedding, enabling us to profile detailed expression kinetics of the earliest blood transcriptional response to contemporaneous variants of concern. 51% of the genes assessed were found to be temporally dynamic during COVID-19 infection. During the pre-shedding phase, a robust but transient response consisting of genes involved in cell migration, stress response, and T cell activation were observed. This is followed by a rapid induction of many interferon-stimulated genes (ISGs), concurrent to onset of viral shedding and increase in nasal viral load and symptom burden. Finally, elevated baseline expression of antimicrobial peptides were observed in exposed-uninfected individuals., Interpretation: We demonstrated that unsupervised self-collection and stabilization of capillary blood can be applied to natural infection studies to characterize detailed early host immune kinetics at a temporal resolution comparable to that of human challenge studies. The remote (decentralized) study framework enables conduct of large-scale population-wide longitudinal mechanistic studies., Funding: This study was funded by R35GM128648 to ABT for in-lab developments of home RNA and data analysis, a Packard Fellowship for Science and Engineering from the David and Lucile Packard Foundation to ABT, and R01AI153087 to AW.

Remote research studies are an invaluable tool for reaching populations in geographical regions with limited access to large medical centers or universities. To expand the remote study toolkit, we have previously developed homeRNA, which allows for at-home self-collection and stabilization of blood and demonstrated the feasibility of using homeRNA in high temperature climates. Here, we expand upon this work through a systematic study exploring the effects of high temperature on RNA integrity through in-lab and field experiments. Compared to the frozen controls (overall mean RIN of 8.2, n = 8), samples kept at 37°C for 2, 4, and 8 days had mean RINs of 7.6, 5.9, and 5.2 ( n = 3), respectively, indicating that typical shipping conditions (~2 days) yield samples suitable for downstream RNA sequencing. Shorter time intervals (6 hours) resulted in minimal RNA degradation (median RIN of 6.4, n = 3) even at higher temperatures (50°C) compared to the frozen control (mean RIN of 7.8, n = 3). Additionally, we shipped homeRNA-stabilized blood from a single donor to 14 different states and back during the summer with continuous temperature probes (7.1 median RIN, n = 42). Samples from all locations were analyzed with 3' mRNA-seq to assess differences in gene counts, with the transcriptomic data suggesting that there was no preferential degradation of transcripts as a result of different shipping times, temperatures, and regions. Overall, our data support that homeRNA can be used in elevated temperature conditions, enabling decentralized sample collection for telemedicine, global health, and clinical research., Competing Interests: Conflicts of Interest EB, AJH, and ABT filed patent 17/361,322 (Publication Number: US20210402406A1) and EB, AJH, ABT, and FS filed patent 63/571,012 through the University of Washington on homeRNA and a related technology. ABT reports filing multiple patents through the University of Washington and receiving a gift to support research outside the submitted work from Ionis Pharmaceuticals. EB has ownership in Salus Discovery, LLC, and Tasso, Inc. that develops blood collection systems used in this manuscript, and is employed by Tasso, Inc. Technologies from Salus Discovery, LLC are not included in this publication. He is an inventor on multiple patents filed by Tasso, Inc., the University of Washington, and the University of Wisconsin-Madison. The terms of this arrangement have been reviewed and approved by the University of Washington in accordance with its policies governing outside work and financial conflicts of interest in research. LGB, SN, and AJH have also filed additional patents through the University of Washington outside the scope of this publication.