A portable surface plasmon resonance (SPR) sensor for the detection of immunoglobulin A in plasma.

BACKGROUND: A life-threatening anaphylactic shock can occur if a patient with undiagnosed immunoglobulin A (IgA) deficiency (i.e., IgA levels <500 ng/mL) receives IgA-containing blood, hence the need for a rapid, point-of-care (POC) method for IgA deficiency screening. Enzyme-linked immunosorbent assay (ELISA) is routinely used to detect IgA, but this method requires trained specialists and ≥24 h to obtain a result. We developed a surface plasmon resonance (SPR)-based protocol to identify IgA-deficient patients or donors within 1 h. MATERIALS AND METHODS: The SPR sensor relies on the detection of IgAs captured by primary antibodies adsorbed on the SPR chip and quantified with secondary antibodies. The sensor was calibrated from 0 to 2000 ng/mL in buffer, IgA-depleted human serum, and plasma samples from IgA-deficient individuals. A critical concentration of 500 ng/mL was set for IgA deficiency. The optimized sensor was then tested on eight plasma samples with known IgA status (determined by ELISA), including five with IgA deficiency and three with normal IgA levels. RESULTS: The limit of detection was estimated at 30 ng/mL in buffer and 400 ng/mL in diluted plasma. The results obtained fully agreed with ELISA among the eight plasma samples tested. The protocol distinguished IgA-deficient from normal samples, even for samples with an IgA concentration closer to critical concentration. DISCUSSION: In conclusion, we developed a reliable POC assay for the quantification of IgA in plasma. This test may permit POC testing at blood drives and centralized centers to maintain reserves of IgA-deficient blood and in-hospital testing of blood recipients.

Monte-Carlo simulation and tissue-phantom model for validation of ocular oximetry.

Ocular oximetry, in which blood oxygen saturation is evaluated in retinal tissues, is a promising technique for the prevention, diagnosis and management of many diseases and conditions. However, the development of new tools for evaluating oxygen saturation in the eye fundus has often been limited by the lack of reference tools or techniques for such measurements. In this study, we describe a two-step validation method. The impact of scattering, blood volume fraction and lens yellowing on the oximetry model is investigated using a tissue phantom, while a Monte Carlo model of the light propagation in the eye fundus is used to study the effect of the fundus layered-structure. With this method, we were able to assess the performance of an ocular oximetry technique in the presence of confounding factors and to quantify the impact of the choroidal circulation on the accuracy of the measurements. The presented strategy will be useful to anyone involved in studies based on the eye fundus diffuse reflectance.

Characterization of the Antibacterial Activity of an SiO2 Nanoparticular Coating to Prevent Bacterial Contamination in Blood Products.

Technological innovations and quality control processes within blood supply organizations have significantly improved blood safety for both donors and recipients. Nevertheless, the risk of transfusion-transmitted infection remains non-negligible. Applying a nanoparticular, antibacterial coating at the surface of medical devices is a promising strategy to prevent the spread of infections. In this study, we characterized the antibacterial activity of an SiO2 nanoparticular coating (i.e., the "Medical Antibacterial and Antiadhesive Coating" [MAAC]) applied on relevant polymeric materials (PM) used in the biomedical field. Electron microscopy revealed a smoother surface for the MAAC-treated PM compared to the reference, suggesting antiadhesive properties. The antibacterial activity was tested against selected Gram-positive and Gram-negative bacteria in accordance with ISO 22196. Bacterial growth was significantly reduced for the MAAC-treated PVC, plasticized PVC, polyurethane and silicone (90-99.999%) in which antibacterial activity of ≥1 log reduction was reached for all bacterial strains tested. Cytotoxicity was evaluated following ISO 10993-5 guidelines and L929 cell viability was calculated at ≥90% in the presence of MAAC. This study demonstrates that the MAAC could prevent bacterial contamination as demonstrated by the ISO 22196 tests, while further work needs to be done to improve the coating processability and effectiveness of more complex matrices.

Assessment of the longitudinal humoral response in non-hospitalized SARS-CoV-2-positive individuals at decentralized sites: Outcomes and concordance.

Introduction: Early in the COVID-19 pandemic, reagent availability was not uniform, and infrastructure had to be urgently adapted to undertake COVID-19 surveillance. Methods: Before the validation of centralized testing, two enzyme-linked immunosorbent assays (ELISA) were established independently at two decentralized sites using different reagents and instrumentation. We compared the results of these assays to assess the longitudinal humoral response of SARS-CoV-2-positive (i.e., PCR-confirmed), non-hospitalized individuals with mild to moderate symptoms, who had contracted SARSCoV-2 prior to the appearance of variants of concern in Québec, Canada. Results: The two assays exhibited a high degree of concordance to identify seropositive individuals, thus validating the robustness of the methods. The results also confirmed that serum immunoglobulins persist ≥ 6 months post-infection among non-hospitalized adults and that the antibodies elicited by infection cross-reacted with the antigens from P.1 (Gamma) and B.1.617.2 (Delta) variants of concern. Discussion: Together, these results demonstrate that immune surveillance assays can be rapidly and reliably established when centralized testing is not available or not yet validated, allowing for robust immune surveillance.

Cross-reactivity of antibodies from non-hospitalized COVID-19 positive individuals against the native, B.1.351, B.1.617.2, and P.1 SARS-CoV-2 spike proteins.

SARS-CoV-2 variants of concern (VOCs) have emerged worldwide, with implications on the spread of the pandemic. Characterizing the cross-reactivity of antibodies against these VOCs is necessary to understand the humoral response of non-hospitalized individuals previously infected with SARS-CoV-2, a population that remains understudied. Thirty-two SARS-CoV-2-positive (PCR-confirmed) and non-hospitalized Canadian adults were enrolled 14-21 days post-diagnosis in 2020, before the emergence of the B.1.351 (also known as Beta), B.1.617.2 (Delta) and P.1 (Gamma) VOCs. Sera were collected 4 and 16 weeks post-diagnosis. Antibody levels and pseudo-neutralization of the ectodomain of SARS-CoV-2 spike protein/human ACE-2 receptor interaction were analyzed with native, B.1.351, B.1.617.2 and P.1 variant spike proteins. Despite a lower response observed for the variant spike proteins, we report evidence of a sustained humoral response against native, B.1.351, B.1.617.2 and P.1 variant spike proteins among non-hospitalized Canadian adults. Furthermore, this response inhibited the interaction between the spike proteins from the different VOCs and ACE-2 receptor for ≥ 16 weeks post-diagnosis, except for individuals aged 18-49 years who showed no inhibition of the interaction between B.1.617.1 or B.1.617.2 spike and ACE-2. Interestingly, the affinity (KD) measured between the spike proteins (native, B.1.351, B.1.617.2 and P.1) and antibodies elicited in sera of infected and vaccinated (BNT162b2 and ChAdOx1 nCoV-19) individuals was invariant. Relative to sera from vaccine-naïve (and previously infected) individuals, sera from vaccinated individuals had higher antibody levels (as measured with label-free SPR) and more efficiently inhibited the spike-ACE-2 interactions, even among individuals aged 18-49 years, showing the effectiveness of vaccination.

Cross-validation of ELISA and a portable surface plasmon resonance instrument for IgG antibody serology with SARS-CoV-2 positive individuals.

We report on the development of surface plasmon resonance (SPR) sensors and matching ELISAs for the detection of nucleocapsid and spike antibodies specific against the novel coronavirus 2019 (SARS-CoV-2) in human serum, plasma and dried blood spots (DBS). When exposed to SARS-CoV-2 or a vaccine against SARS-CoV-2, the immune system responds by expressing antibodies at levels that can be detected and monitored to identify the fraction of the population potentially immunized against SARS-CoV-2 and support efforts to deploy a vaccine strategically. A SPR sensor coated with a peptide monolayer and functionalized with various sources of SARS-CoV-2 recombinant proteins expressed in different cell lines detected human anti-SARS-CoV-2 IgG antibodies in clinical samples. Nucleocapsid expressed in different cell lines did not significantly change the sensitivity of the assays, whereas the use of a CHO cell line to express spike ectodomain led to excellent performance. This bioassay was performed on a portable SPR instrument capable of measuring 4 biological samples within 30 minutes of sample/sensor contact and the chip could be regenerated at least 9 times. Multi-site validation was then performed with in-house and commercial ELISA, which revealed excellent cross-correlations with Pearson's coefficients exceeding 0.85 in all cases, for measurements in DBS and plasma. This strategy paves the way to point-of-care and rapid testing for antibodies in the context of viral infection and vaccine efficacy monitoring.

Drone versus ground delivery of simulated blood products to an urban trauma center: The Montreal Medi-Drone pilot study.

BACKGROUND: Timely and safe distribution of quality blood products is a major challenge faced by blood banks around the world. Our primary objective was to determine if simulated blood product delivery to an urban trauma center would be more rapidly achieved by unmanned aerial vehicle (UAV) than by ground transportation. A secondary objective was to determine the feasibility of maintaining simulated blood product temperatures within a targeted range. METHODS: In this prospective pilot study, we used two distinct methods to compare UAV flight duration and ground transport times. Simulated blood products included packed red blood cells, platelet concentrate, and fresh frozen plasma. For each blood product type, three UAV flights were conducted. Temperature was monitored during transport using a probe coupled to a data logger inside each simulated blood product unit. RESULTS: All flights were conducted successfully without any adverse events or safety concerns reported. The heaviest payload transported was 6.4 kg, and the drone speed throughout all nine flights was 10 m/s. The mean UAV transportation time was significantly faster than ground delivery (17:06 ± 00:04 minutes vs. 28:54 ± 01:12 minutes, p < 0.0001). The mean ± SD initial temperature for packed red blood cells was 4.4°C ± 0.1°C with a maximum 5% mean temperature variability from departure to landing. For platelet concentrates, the mean ± SD initial temperature was 21.6°C ± 0.5°C, and the maximum variability observed was 0.3%. The mean ± SD initial fresh frozen plasma temperature was -19°C ± 2°C, and the greatest temperature variability was from -17°C ± 2°C to -16°C ± 2°C. CONCLUSIONS: Unmanned aerial vehicle transportation of simulated blood products was significantly faster than ground delivery. Simulated blood product temperatures remained within their respective acceptable ranges throughout transport. Further studies assessing UAV transport of real blood products in populated areas are warranted. LEVEL OF EVIDENCE: Therapeutic/care management, level IV.

Evaluation of the Tempo® System: Improving the Microbiological Quality Monitoring of Human Milk.

Background: Bacteriological testing of donor human milk is mostly done both before and after pasteurization to control contamination in the end-product and meet the microbiological standards. Although the plate count method represents a reliable and sensitive technique and is considered the gold standard for bacteriological testing, it is recognized for being time-consuming and requiring qualified personnel. Recently, faster testing technologies, mostly geared toward the food industry, have been developed. Among these, the bioMérieux TEMPO® system uses the most probable number method to assess microbiological content in a semi-automated fashion. Objective: The performances of the TEMPO® system in enumerating bacterial quality indicators in human milk were assessed and compared to the reference plate count method. Methods: Naturally and artificially contaminated human milk samples were used to compare the analytical performances of the TEMPO® system to the plate count technique. More specifically, bacteria belonging to the genera Bacillus, Enterobacteriaceae, Staphylococcus aureus, and total aerobic flora were screened using both methods. Bacteria isolated on agar plates containing selective media were identified by supplemental testing. Bacterial testing results and method parameters were compared using linear regression analyses and Bland-Altman approaches. Results: Naturally contaminated milk samples (n = 55) tested for total aerobic flora showed < 1 log (CFU/ml) discrepancy between the two methods in the output results for 98% of the samples. Comparative linear regression analyses demonstrate good correlations between the two methods (R 2 > 0.9). At lower levels of bacterial contamination, the TEMPO® method precision (C.V. < 8%) and accuracy (> 83%) were comparable to plate counts. Conclusions: The analytical performances of the TEMPO® system for human milk bacteriological testing are equivalent to the reference plate count method. Results from the TEMPO® system are available within a 24-h turnaround time from sample inoculation without the need for further supplemental testing, suggesting that this semi-automated method could be implemented within milk bank operations as an in-process monitoring technology to optimize end-product quality and safety.

Development of a flow standard to enable highly reproducible measurements of deformability of stored red blood cells in a microfluidic device.

BACKGROUND: Great deformability allows red blood cells (RBCs) to flow through narrow capillaries in tissues. A number of microfluidic devices with capillary-like microchannels have been developed to monitor storage-related impairment of RBC deformability during blood banking operations. This proof-of-concept study describes a new method to standardize and improve reproducibility of the RBC deformability measurements using one of these devices. STUDY DESIGN AND METHODS: The rate of RBC flow through the microfluidic capillary network of the microvascular analyzer (MVA) device made of polydimethylsiloxane was measured to assess RBC deformability. A suspension of microbeads in a solution of glycerol in phosphate-buffered saline was developed to be used as an internal flow rate reference alongside RBC samples in the same device. RBC deformability and other in vitro quality markers were assessed weekly in six leukoreduced RBC concentrates (RCCs) dispersed in saline-adenine-glucose-mannitol additive solution and stored over 42 days at 4°C. RESULTS: The use of flow reference reduced device-to-device measurement variability from 10% to 2%. Repeated-measure analysis using the generalized estimating equation (GEE) method showed a significant monotonic decrease in relative RBC flow rate with storage from Week 0. By the end of storage, relative RBC flow rate decreased by 22 ± 6% on average. CONCLUSIONS: The suspension of microbeads was successfully used as a flow reference to increase reproducibility of RBC deformability measurements using the MVA. Deformability results suggest an early and late aging phase for stored RCCs, with significant decreases between successive weeks suggesting a highly sensitive measurement method.

Internalization of targeted quantum dots by brain capillary endothelial cells in vivo.

Receptors located on brain capillary endothelial cells forming the blood-brain barrier are the target of most brain drug delivery approaches. Yet, direct subcellular evidence of vectorized transport of nanoformulations into the brain is lacking. To resolve this question, quantum dots were conjugated to monoclonal antibodies (Ri7) targeting the murine transferrin receptor. Specific transferrin receptor-mediated endocytosis of Ri7-quantum dots was first confirmed in N2A and bEnd5 cells. After intravenous injection in mice, Ri7-quantum dots exhibited a fourfold higher volume of distribution in brain tissues, compared to controls. Immunofluorescence analysis showed that Ri7-quantum dots were sequestered throughout the cerebral vasculature 30 min, 1 h, and 4 h post injection, with a decline of signal intensity after 24 h. Transmission electron microscopic studies confirmed that Ri7-quantum dots were massively internalized by brain capillary endothelial cells, averaging 37 ± 4 Ri7-quantum dots/cell 1 h after injection. Most quantum dots within brain capillary endothelial cells were observed in small vesicles (58%), with a smaller proportion detected in tubular structures or in multivesicular bodies. Parenchymal penetration of Ri7-quantum dots was extremely low and comparable to control IgG. Our results show that systemically administered Ri7-quantum dots complexes undergo extensive endocytosis by brain capillary endothelial cells and open the door for novel therapeutic approaches based on brain endothelial cell drug delivery.

Label-free biosensing based on multilayer fluorescent nanocomposites and a cationic polymeric transducer.

This study describes the preparation and characterization of a DNA sensing architecture combining the molecular recognition capabilities of a cationic conjugated polymer transducer with highly fluorescent core-shell nanoparticles (NPs). The very structure of the probe-labeled NPs and the polymer-induced formation of NP aggregates maximize the proximity between the polymer donor and acceptor NPs that is required for optimal resonant energy transfer. Each hybridization event is signaled by a potentially large number of excited reporters following the efficient plasmon-enhanced energy transfer between target-activated polymer transducer and fluorophores located in the self-assembled core-shell aggregates, resulting in direct molecular detection of target nucleic acids at femtomolar concentrations.

Use of sol-gels as solid matrixes for trace analysis by UV laser ablation and laser-enhanced ionization detection.

Zirconium and silicon sol-gels were investigated as solid materials for trace elemental analysis of pelletized solid samples by laser ablation and laser-enhanced ionization. The highly homogeneous dispersion of an internal standard spiked in the solid material obtained with the sol-gel formation process leads to a significant improvement in signal repeatability and to an increase in the precision of measurements through better correction of variations in the laser ablation rate. Signal repeatability values of 5-8% RSD were obtained for Pb in NIST 1632c Bituminous Coal sample pellets prepared using both sol-gels, as compared to 9-21% for graphite-based sample pellets. Furthermore, the zirconium sol-gel was shown to offer better resilience to signal bias due to preferential ablation and a more accurate correction of ablation rate using the internal standardization method.