A Possible Way to Relate the Effects of SARS-CoV-2-Induced Changes in Transferrin to Severe COVID-19-Associated Diseases.

SARS-CoV-2 infections are responsible for the COVID-19 pandemic. Transferrin has been found to explain the link between diseases associated with impaired iron transport and COVID-19 infection. The effect of SARS-CoV-2 on human whole blood was studied by differential scanning calorimetry. The analysis of the thermal transition curves showed that the melting temperature of the transferrin-related peak decreased in the presence of SARS-CoV-2. The ratio of the under-curve area of the two main peaks was greatly affected, while the total enthalpy of the heat denaturation remained nearly unchanged in the presence of the virus. These results indicate that SARS-CoV-2, through binding to transferrin, may influence its Fe3+ uptake by inducing thermodynamic changes. Therefore, transferrin may remain in an iron-free apo-conformational state, which depends on the SARS-CoV-2 concentration. SARS-CoV-2 can induce disturbance in erythropoiesis due to toxicity generated by free iron overload.

A streamlined whole blood CyTOF workflow defines a circulating immune cell signature of COVID-19.

Mass cytometry (CyTOF) represents one of the most powerful tools in immune phenotyping, allowing high throughput quantification of over 40 parameters at single-cell resolution. However, wide deployment of CyTOF-based immune phenotyping studies are limited by complex experimental workflows and the need for specialized CyTOF equipment and technical expertise. Furthermore, differences in cell isolation and enrichment protocols, antibody reagent preparation, sample staining, and data acquisition protocols can all introduce technical variation that can confound integrative analyses of large data-sets of samples processed across multiple labs. Here, we present a streamlined whole blood CyTOF workflow which addresses many of these sources of experimental variation and facilitates wider adoption of CyTOF immune monitoring across sites with limited technical expertise or sample-processing resources or equipment. Our workflow utilizes commercially available reagents including the Fluidigm MaxPar Direct Immune Profiling Assay (MDIPA), a dry tube 30-marker immunophenotyping panel, and SmartTube Proteomic Stabilizer, which allows for simple and reliable fixation and cryopreservation of whole blood samples. We validate a workflow that allows for streamlined staining of whole blood samples with minimal processing requirements or expertise at the site of sample collection, followed by shipment to a central CyTOF core facility for batched downstream processing and data acquisition. We apply this workflow to characterize 184 whole blood samples collected longitudinally from a cohort of 72 hospitalized COVID-19 patients and healthy controls, highlighting dynamic disease-associated changes in circulating immune cell frequency and phenotype.

Validation of a liquid chromatography tandem mass spectrometry method for the simultaneous determination of hydroxychloroquine and metabolites in human whole blood.

Objectives Hydroxychloroquine (HCQ) is an anti-malarial and immunomodulatory drug reported to inhibit the Corona virus, SARS-CoV-2, in vitro. At present there is insufficient evidence from clinical trials to determine the safety and efficacy of HCQ as a treatment for COVID-19. However, since the World Health Organisation declared COVID-19 a pandemic in March 2020, the US Food and Drug Administration issued an Emergency Use Authorisation to allow HCQ and Chloroquine (CQ) to be distributed and used for certain hospitalised patients with COVID-19 and numerous clinical trials are underway around the world, including the UK based RECOVERY trial, with over 1000 volunteers. The validation of a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of HCQ and two of its major metabolites, desethylchloroquine (DCQ) and di-desethylchloroquine (DDCQ), in whole blood is described. Methods Blood samples were deproteinised using acetonitrile. HCQ, DCQ and DDCQ were chromatographically separated on a biphenyl column with gradient elution, at a flow rate of 500 µL/min. The analysis time was 8 min. Results For each analyte linear calibration curves were obtained over the concentration range 50-2000 µg/L, the lower limit of quantification (LLOQ) was 13 µg/L, the inter-assay relative standard deviation (RSD) was <10% at 25, 800 and 1750 µg/L and mean recoveries were 80, 81, 78 and 62% for HCQ, d4-HCQ, DCQ and DDCQ, respectively. Conclusion This method has acceptable analytical performance and is applicable to the therapeutic monitoring of HCQ, evaluating the pharmacokinetics of HCQ in COVID-19 patients and supporting clinical trials.