Clinical Validation of a Novel T-Cell Receptor Sequencing Assay for Identification of Recent or Prior Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

BACKGROUND: While diagnostic, therapeutic, and vaccine development in the coronavirus disease 2019 (COVID-19) pandemic has proceeded at unprecedented speed, critical gaps in our understanding of the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain unaddressed by current diagnostic strategies. METHODS: A statistical classifier for identifying prior SARS-CoV-2 infection was trained using >4000 SARS-CoV-2-associated T-cell receptor (TCR) ß sequences identified by comparing 784 cases and 2447 controls from 5 independent cohorts. The T-Detect COVID (Adaptive Biotechnologies) assay applies this classifier to TCR repertoires sequenced from blood samples to yield a binary assessment of past infection. Assay performance was assessed in 2 retrospective (n = 346; n = 69) and 1 prospective cohort (n = 87) to determine positive percent agreement (PPA) and negative percent agreement (NPA). PPA was compared with 2 commercial serology assays, and pathogen cross-reactivity was evaluated. RESULTS: T-Detect COVID demonstrated high PPA in individuals with prior reverse transcription-polymerase chain reaction (RT-PCR)-confirmed SARS-CoV-2 infection (97.1% 15+ days from diagnosis; 94.5% 15+ days from symptom onset), high NPA (∼100%) in presumed or confirmed SARS-CoV-2 negative cases, equivalent or higher PPA than 2 commercial serology tests, and no evidence of pathogen cross-reactivity. CONCLUSIONS: T-Detect COVID is a novel T-cell immunosequencing assay demonstrating high clinical performance for identification of recent or prior SARS-CoV-2 infection from blood samples, with implications for clinical management, risk stratification, surveillance, and understanding of protective immunity and long-term sequelae.

Development of Gene Expression-Based Biomarkers on the nCounter® Platform for Immuno-Oncology Applications.

Biomarkers based on transcriptional profiling can be useful in the measurement of complex and/or dynamic physiological states where other profiling strategies such as genomic or proteomic characterization are not able to adequately measure the biology. One particular advantage of transcriptional biomarkers is the ease with which they can be measured in the clinical setting using robust platforms such as the NanoString nCounter system. The nCounter platform enables digital quantitation of multiplexed RNA from small amounts of blood, formalin-fixed, paraffin-embedded tumors, or other such biological samples that are readily available from patients, and the chapter uses it as the primary example for diagnostic assay development. However, development of diagnostic assays based on RNA biomarkers on any platform requires careful consideration of all aspects of the final clinical assay a priori, as well as design and execution of the development program in a way that will maximize likelihood of future success. This chapter introduces transcriptional biomarkers and provides an overview of the design and development process that will lead to a locked diagnostic assay that is ready for validation of clinical utility.

T2 magnetic resonance: a diagnostic platform for studying integrated hemostasis in whole blood--proof of concept.

BACKGROUND: Existing approaches for measuring hemostasis parameters require multiple platforms, can take hours to provide results, and generally require 1-25 mL of sample. We developed a diagnostic platform that allows comprehensive assessment of hemostatic parameters on a single instrument and provides results within 15 min using 0.04 mL of blood with minimal sample handling. METHODS: T2 magnetic resonance (T2MR) was used to directly measure integrated reactions in whole blood samples by resolving multiple water relaxation times from distinct sample microenvironments. Clotting, clot contraction, and fibrinolysis stimulated by thrombin or tissue plasminogen activator, respectively, were measured. T2MR signals of clotting samples were compared with images produced by scanning electron microscopy and with standard reference methods for the following parameters: hematocrit, prothrombin time, clot strength, and platelet activity. RESULTS: Application of T2MR methodology revealed conditions under which a unique T2MR signature appeared that corresponded with the formation of polyhedral erythrocytes, the dynamics and morphology of which are dependent on thrombin, fibrinogen, hematocrit, and platelet levels. We also showed that the T2MR platform can be used for precise and accurate measurements of hematocrit (%CV, 4.8%, R(2) = 0.95), clotting time (%CV, 3.5%, R(2) = 0.94), clot strength (R(2) = 0.95), and platelet function (93% agreement with light transmission aggregometry). CONCLUSIONS: This proof-of-concept study demonstrates that T2MR has the potential to provide rapid and sensitive identification of patients at risk for thrombosis or bleeding and to identify new biomarkers and therapeutic targets with a single, simple-to-employ analytic approach that may be suitable for routine use in both research and diverse clinical settings.

T2 magnetic resonance enables nanoparticle-mediated rapid detection of candidemia in whole blood.

Candida spp. cause both local and disseminated infections in immunocompromised patients. Bloodstream infections of Candida spp., known as "candidemia," are associated with a high mortality rate (40%), which is mainly attributed to the long diagnostic time required by blood culture. We introduce a diagnostic platform based on T2 magnetic resonance (T2MR), which is capable of sensitive and rapid detection of fungal targets in whole blood. In our approach, blood-compatible polymerase chain reaction is followed by hybridization of the amplified pathogen DNA to capture probe-decorated nanoparticles. Hybridization yields nanoparticle microclusters that cause large changes in the sample's T2MR signal. With this T2MR-based method, Candida spp. can be detected directly in whole blood, thus eliminating the need for analyte purification. Using a small, portable T2MR detection device, we were able to rapidly, accurately, and reproducibly detect five Candida species within human whole blood with a limit of detection of 1 colony-forming unit/ml and a time to result of <3 hours. Spiked blood samples showed 98% positive agreement and 100% negative agreement between T2MR and blood culture. Additionally, performance of the assay was evaluated on 21 blinded clinical specimens collected serially. This study shows that the nanoparticle- and T2MR-based detection method is rapid and amenable to automation and offers clinicians the opportunity to detect and identify multiple human pathogens within hours of sample collection.

Control of colloid surface chemistry through matrix confinement: facile preparation of stable antibody functionalized silver nanoparticles.

Here we describe a simple yet efficient gel-matrix-assisted preparation method that improves synthetic control over the interface between inorganic nanomaterials and biopolymers and yields stable biofunctionalized silver nanoparticles. Covalent functionalization of the noble metal surface is aided by the confinement of polyethylene glycol acetic acid functionalized silver nanoparticles in thin slabs of a 1% agarose gel. The gel-confined nanoparticles can be transferred between reaction and washing media simply by immersing the gel slab in the solution of interest. The agarose matrix retains nanoparticles but is swiftly penetrated by the antibodies of interest. The antibodies are covalently anchored to the nanoparticles using conventional cross-linking strategies, and the resulting antibody functionalized nanoparticles are recovered from the gel through electroelution. We demonstrate the efficacy of this nanoparticle functionalization approach by labeling specific receptors on cellular surfaces with functionalized silver nanoparticles that are stable under physiological conditions.

Resolving sub-diffraction limit encounters in nanoparticle tracking using live cell plasmon coupling microscopy.

We use plasmon coupling between individual gold nanoparticle labels to monitor subdiffraction limit distances in live cell nanoparticle tracking experiments. While the resolving power of our optical microscope is limited to approximately 500 nm, we improve this by more than an order of magnitude by detecting plasmon coupling between individual gold nanoparticle labels using a ratiometric detection scheme. We apply this plasmon coupling microscopy to resolve the interparticle separations during individual encounters of gold nanoparticle labeled fibronectin-integrin complexes in living HeLa cells.

Spermidine modulated ribonuclease activity probed by RNA plasmon rulers.

We extend the scope of nanometer distance measurements based on coupled pairs of gold nanoparticles, or plasmon rulers, to individual RNA molecules. These sensors were used to monitor the influence of spermidine on the cleavage kinetics of RNA by ribonuclease A. Time-resolved cleavage experiments of individual RNA plasmon rulers reveal transiently stabilized RNA sub-populations at increased spermidine concentrations that indicate spermidine-induced stabilization of weak secondary and tertiary structural elements.