Development of a Paper-based Hematocrit Test and a Lateral Flow Assay to Detect Critical Fibrinogen Concentrations Using a Bottom-Up Pyramid Workflow Approach.

Fibrinogen is a coagulation factor in human blood and the first one to reach critical levels in major bleeding. Hypofibrinogenemia (a too low fibrinogen concentration in blood) poses great challenges to first responders, clinicians, and healthcare providers since it represents a risk factor for exsanguination and massive transfusion requirements. Thus, the rapid assessment of the fibrinogen concentration at the point of care has gained considerable importance in preventing and managing major blood loss. However, in whole blood measurements, hematocrit variations affect the amount (volume fraction) of plasma that passes the detection zone. In an attempt to accurately determine realistic critical levels of fibrinogen (<1.5 mg/mL) in patients needing immediate treatment and medical interventions, we have developed novel diagnostic systems capable of estimating hematocrit and critical fibrinogen concentrations. A lateral flow assay (LFA) for the detection of fibrinogen has been developed by establishing a workflow employing rapid characterization methods to streamline LFA development. The integration of two detection lines enables (i) the identification of fibrinogen (first line) present in the sample and (ii) the determination of the clinically critical fibrinogen concentrations below 1.5 mg/mL (second line). Furthermore, the paper-based separation of blood cells from plasma provides a semiquantitative estimate of the hematocrit by analyzing the fractions. Initial validation of the point-of-care (PoC) hematocrit test revealed good comparability to a standard laboratory method. The developed diagnostic systems have the ability to accelerate decision-making in cases with major bleeding.

Application of a Biomimetic Nanoparticle-Based Mock Virus to Determine SARS-CoV-2 Neutralizing Antibody Levels in Blood Samples Using a Lateral Flow Assay.

The presence of neutralizing antibodies against SARS-CoV-2 in blood, acquired through previous infection or vaccination, is known to prevent the (re)occurrence of outbreaks unless the virus mutates. Therefore, the measurement of neutralizing antibodies constitutes an indispensable tool in assessing an individual's and a population's immunity against SARS-CoV-2. For this reason, we have developed an innovative lateral flow assay (LFA) capable of detecting blood-derived neutralizing antibodies using a biomimetic SARS-CoV-2 mock virus system. Here, functionalized gold nanoparticles (AuNPs) featuring the trimeric spike (S) protein at its surface imitate the virus's structure and are applied to monitor the presence and efficacy of neutralizing antibodies in blood samples. The detection principle relies on the interaction between mock virus and the immobilized angiotensin-converting enzyme 2 (ACE2) receptor, which is inhibited when neutralizing antibodies are present. To further enhance the sensitivity of our competitive assay and identify low titers of neutralizing antibodies, an additional mixing pad is embedded into the device to increase the interaction time between mock virus and neutralizing antibodies. The developed LFA is benchmarked against the WHO International Standard (21/338) and demonstrated reliable quantification of neutralizing antibodies that inhibit ACE2 binding events down to a detection limit of an antibody titer of 59 IU/mL. Additional validation using whole blood and plasma samples showed reproducible results and good comparability to a laboratory-based reference test, thus highlighting its applicability for point-of-care testing.

Circulating endothelial extracellular vesicle signatures correspond with ICU requirement: an exploratory study in COVID-19 patients.

Extracellular vesicles (EVs) represent nanometer-sized, subcellular spheres, that are released from almost any cell type and carry a wide variety of biologically relevant cargo. In severe cases of coronavirus disease 2019 (COVID-19) and other states of systemic pro-inflammatory activation, EVs, and their cargo can serve as conveyors and indicators for disease severity and progression. This information may help distinguish individuals with a less severe manifestation of the disease from patients who exhibit severe acute respiratory distress syndrome (ARDS) and require intensive care measures. Here, we investigated the potential of EVs and associated miRNAs to distinguish normal ward patients from intensive care unit (ICU) patients (N = 10/group), with 10 healthy donors serving as the control group. Blood samples from which plasma and subsequently EVs were harvested by differential ultracentrifugation (UC) were obtained at several points in time throughout treatment. EV-enriched fractions were characterized by flow cytometry (FC), nanoparticle tracking analysis (NTA), and qPCR to determine the presence of selected miRNAs. Circulating EVs showed specific protein signatures associated with endothelial and platelet origin over the course of the treatment. Additionally, significantly higher overall EV quantities corresponded with increased COVID-19 severity. MiR-223-3p, miR-191-5p, and miR-126-3p exhibited higher relative expression in the ICU group. Furthermore, EVs presenting endothelial-like protein signatures and the associated miR-126-3p showed the highest area under the curve in terms of receiver operating characteristics regarding the requirement for ICU treatment. In this exploratory investigation, we report that specific circulating EVs and miRNAs appear at higher levels in COVID-19 patients, especially when critical care measures are indicated. Our data suggest that endothelial-like EVs and associated miRNAs likely represent targets for future laboratory assays and may aid in clinical decision-making in COVID-19.