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1.
Adv Sci (Weinh) ; 8(18): e2100323, 2021 09.
Article in English | MEDLINE | ID: covidwho-1316190

ABSTRACT

Blood cell analysis is a major pillar of biomedical research and healthcare. These analyses are performed in central laboratories. Rapid shipment from collection site to the central laboratories is currently needed because cells and biomarkers degrade rapidly. The dried blood spot from a fingerstick allows the preservation of cellular molecules for months but entire cells are never recovered. Here leucocyte elution is optimized from dried blood spots. Flow cytometry and mRNA expression profiling are used to analyze the recovered cells. 50-70% of the leucocytes that are dried on a polyester solid support via elution after shaking the support with buffer are recovered. While red blood cells lyse upon drying, it is found that the majority of leucocytes are preserved. Leucocytes have an altered structure that is improved by adding fixative in the elution buffer. Leucocytes are permeabilized, allowing an easy staining of all cellular compartments. Common immunophenotyping and mRNAs are preserved. The ability of a new biomarker (CD169) to discriminate between patients with and without Severe Acute Respiratory Syndrome induced by Coronavirus 2 (SARS-CoV-2) infections is also preserved. Leucocytes from blood can be dried, shipped, and/or stored for at least 1 month, then recovered for a wide variety of analyses, potentially facilitating biomedical applications worldwide.


Subject(s)
Communicable Diseases/diagnosis , Diagnostic Tests, Routine/methods , Dried Blood Spot Testing/methods , Hematology/methods , Immunophenotyping/methods , Antibodies, Viral/blood , Biomarkers/blood , Blood Specimen Collection/methods , COVID-19/diagnosis , Cell Separation/methods , Communicable Diseases/virology , Erythrocytes/virology , Flow Cytometry/methods , Humans , Leukocytes/virology , RNA, Messenger/blood , SARS-CoV-2/genetics
2.
Sci Rep ; 11(1): 14015, 2021 07 07.
Article in English | MEDLINE | ID: covidwho-1301182

ABSTRACT

Venous thromboembolism is the third common cardiovascular disease and is composed of two entities, deep vein thrombosis (DVT) and its potential fatal form, pulmonary embolism (PE). While PE is observed in ~ 40% of patients with documented DVT, there is limited biomarkers that can help identifying patients at high PE risk. To fill this need, we implemented a two hidden-layers artificial neural networks (ANN) on 376 antibodies and 19 biological traits measured in the plasma of 1388 DVT patients, with or without PE, of the MARTHA study. We used the LIME algorithm to obtain a linear approximate of the resulting ANN prediction model. As MARTHA patients were typed for genotyping DNA arrays, a genome wide association study (GWAS) was conducted on the LIME estimate. Detected single nucleotide polymorphisms (SNPs) were tested for association with PE risk in MARTHA. Main findings were replicated in the EOVT study composed of 143 PE patients and 196 DVT only patients. The derived ANN model for PE achieved an accuracy of 0.89 and 0.79 in our training and testing sets, respectively. A GWAS on the LIME approximate identified a strong statistical association peak (rs1424597: p = 5.3 × 10-7) at the PLXNA4 locus. Homozygote carriers for the rs1424597-A allele were then more frequently observed in PE than in DVT patients from the MARTHA (2% vs. 0.4%, p = 0.005) and the EOVT (3% vs. 0%, p = 0.013) studies. In a sample of 112 COVID-19 patients known to have endotheliopathy leading to acute lung injury and an increased risk of PE, decreased PLXNA4 levels were associated (p = 0.025) with worsened respiratory function. Using an original integrated proteomics and genetics strategy, we identified PLXNA4 as a new susceptibility gene for PE whose exact role now needs to be further elucidated.


Subject(s)
Genetic Predisposition to Disease/genetics , Neural Networks, Computer , Proteomics , Pulmonary Embolism/blood , Pulmonary Embolism/genetics , Receptors, Cell Surface/blood , Receptors, Cell Surface/genetics , Adult , COVID-19/complications , Female , Genome-Wide Association Study , Humans , Male , Phenotype , Polymorphism, Single Nucleotide , Pulmonary Embolism/complications , Pulmonary Embolism/metabolism
4.
J Infect Dis ; 222(12): 1985-1996, 2020 11 13.
Article in English | MEDLINE | ID: covidwho-1059699

ABSTRACT

BACKGROUND: An unbiased approach to SARS-CoV-2-induced immune dysregulation has not been undertaken so far. We aimed to identify previously unreported immune markers able to discriminate COVID-19 patients from healthy controls and to predict mild and severe disease. METHODS: An observational, prospective, multicentric study was conducted in patients with confirmed mild/moderate (n = 7) and severe (n = 19) COVID-19. Immunophenotyping of whole-blood leukocytes was performed in patients upon hospital ward or intensive care unit admission and in healthy controls (n = 25). Clinically relevant associations were identified through unsupervised analysis. RESULTS: Granulocytic (neutrophil, eosinophil, and basophil) markers were enriched during COVID-19 and discriminated between patients with mild and severe disease. Increased counts of CD15+CD16+ neutrophils, decreased granulocytic expression of integrin CD11b, and Th2-related CRTH2 downregulation in eosinophils and basophils established a COVID-19 signature. Severity was associated with emergence of PD-L1 checkpoint expression in basophils and eosinophils. This granulocytic signature was accompanied by monocyte and lymphocyte immunoparalysis. Correlation with validated clinical scores supported pathophysiological relevance. CONCLUSIONS: Phenotypic markers of circulating granulocytes are strong discriminators between infected and uninfected individuals as well as between severity stages. COVID-19 alters the frequency and functional phenotypes of granulocyte subsets with emergence of CRTH2 as a disease biomarker.


Subject(s)
COVID-19/immunology , Granulocytes/immunology , Receptors, Immunologic/metabolism , Receptors, Prostaglandin/metabolism , Adult , Aged , Biomarkers/metabolism , CD11b Antigen/immunology , COVID-19/blood , COVID-19/diagnosis , Female , France , Humans , Immunophenotyping , Leukocyte Count , Lymphocytes/immunology , Male , Middle Aged , Monocytes/immunology , Prospective Studies , SARS-CoV-2 , Severity of Illness Index
5.
Cytometry A ; 99(5): 435-445, 2021 05.
Article in English | MEDLINE | ID: covidwho-1046850

ABSTRACT

The identification of a bacterial, viral, or even noninfectious cause is essential in the management of febrile syndrome in the emergency department (ED), especially in epidemic contexts such as flu or CoVID-19. The aim was to assess discriminative performances of two biomarkers, CD64 on neutrophils (nCD64) and CD169 on monocytes (mCD169), using a new flow cytometry procedure, in patients presenting with fever to the ED during epidemics. Eighty five adult patients presenting with potential infection were included during the 2019 flu season in the ED of La Timone Hospital. They were divided into four diagnostic outcomes according to their clinical records: no-infection, bacterial infection, viral infection and co-infection. Seventy six patients with confirmed SARS-CoV-2 infection were also compared to 48 healthy volunteers. For the first cohort, 38 (45%) patients were diagnosed with bacterial infections, 11 (13%) with viral infections and 29 (34%) with co-infections. mCD169 was elevated in patients with viral infections, with a majority of Flu A virus or Respiratory Syncytial Virus, while nCD64 was elevated in subjects with bacterial infections, with a majority of Streptococcus pneumoniae and Escherichia coli. nCD64 and mCD169 showed 90% and 80% sensitivity, and 78% and 91% specificity, respectively, for identifying patients with bacterial or viral infections. When studied in a second cohort, mCD169 was elevated in 95% of patients with SARS-CoV-2 infections and remained at normal level in 100% of healthy volunteers. nCD64 and mCD169 have potential for accurately distinguishing bacterial and acute viral infections. Combined in an easy and rapid flow cytometry procedure, they constitute a potential improvement for infection management in the ED, and could even help for triage of patients during emerging epidemics.


Subject(s)
Bacterial Infections/diagnosis , COVID-19/diagnosis , Emergency Service, Hospital , Flow Cytometry , Monocytes/immunology , Receptors, IgG/blood , Sialic Acid Binding Ig-like Lectin 1/blood , Adult , Aged , Bacterial Infections/blood , Bacterial Infections/immunology , Bacterial Infections/microbiology , Biomarkers/blood , COVID-19/blood , COVID-19/immunology , COVID-19/virology , Diagnosis, Differential , Female , Host-Pathogen Interactions , Humans , Male , Middle Aged , Monocytes/microbiology , Monocytes/virology , Predictive Value of Tests , Prospective Studies , Reproducibility of Results
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