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To identify COVID-19-associated immunophenotyping patterns at hospital admission and to determine if some patterns could predict the need for mechanical ventilation (MV)., Design: Prospective observational monocentric cohort study., Setting: A university-affiliated hospital in Marseille, France., Patients: Thirty patients presenting with laboratory-confirmed COVID-19 pneumonia were enrolled within the first 48 hours of hospital admission and compared with 18 healthy controls., Interventions: None., Measurements and Main Results: Whole-blood leukocytes were immunophenotyped with a rapid and simplified one-step flow cytometry method. Thirty-eight immune and five laboratory parameters were compared first between COVID-19 patients and controls and then between the COVID-19 patients who received or not MV during their stays. The variables that significantly discriminated MV from non-MV patients in univariate analysis were entered into a multiple stepwise logistic regression analysis. The COVID-19 patients were predominantly male (87%), aged 61 years (50-71 yr), and 93% received early corticosteroid therapy. Sixteen patients (53%) were managed with noninvasive respiratory support, and 14 (47%) required MV. Compared with controls, COVID-19 patients were characterized by an immune signature featuring: 1) decreased HLA-DR expression on monocytes; 2) reduced basophils, eosinophils, T-cells, NK cells, and nonclassical monocyte count; and 3) up regulation of CD169 on monocytes, CD64 on neutrophils, the adhesion/migration markers (CD62L and CD11b), and the checkpoint inhibitor CD274 on myeloid cells. Among the COVID-19 patients, those who received MV had lower level of CD4 and HLA-DR on monocytes, lower CD8+ T-cell count, and higher lactate dehydrogenase at hospital admission. In multivariate analysis, only CD4 on monocytes ( p = 0.032) and CD8+ T-cell count ( p = 0.026) were associated with MV requirement. The model combining these two variables provided an area under curve of 0.97 (95% CI, 0.83-0.99)., Conclusions: The association of low CD4 on monocytes and low CD8+ T-cell count at hospital admission was highly predictive of the need for MV in hospitalized patients with COVID-19 pneumonia., Competing Interests: The authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2022 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of the Society of Critical Care Medicine.)

A strong bias related to age is observed in COVID-19 patients with pediatric subjects developing a milder disease than adults. We hypothesized that a specific SARS-CoV-2 effect conjugated with preexisting differences in the immune systems may explain this. Using flow cytometry, we investigated basal immune differences in a cohort consisting of 16 non-infected young and 16 aged individuals and further leveraged an in vitro whole blood model of SARS-CoV-2 infection so that functional differences could be mined as well. In short, blood diluted in culture media was incubated 5 or 24 h with the trimeric spike protein or controls. Following unsupervised analysis, we first confirmed that the immune lymphoid and myeloid systems in adults are less efficient and prone to develop higher inflammation than those in children. We notably identified in adults a higher CD43 lymphocyte expression, known for its potentially inhibitory role. The spike protein induced different responses between adults and children, notably a higher increase of inflammatory markers together with lower monocyte and B cell activation in adults. Interestingly, CD169, a CD43 ligand overexpressed in COVID-19 patients, was confirmed to be strongly modulated by the spike protein. In conclusion, the spike protein exacerbated the preexisting lower immune responsiveness and higher inflammatory potential in adults. Altogether, some of the markers identified may explain the marked age bias and be predictive of severity., (© 2022. The Author(s).)

Objective: The COVID-19 corona virus disease outbreak is globally challenging health systems and societies. Its diagnosis relies on molecular methods, with drawbacks revealed by mass screening. Upregulation of neutrophil CD64 or monocyte CD169 has been abundantly reported as markers of bacterial or acute viral infection, respectively. We evaluated the sensitivity of an easy, one-step whole blood flow cytometry assay to measure these markers within 10 min, as a potential screening test for COVID-19 patients., Methods: Patients ( n  = 177) with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were tested on 10 µL blood and results were compared with reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR)., Results: We observed 98% and 100% sensitivity in early-stage ( n  = 52) and asymptomatic patients ( n  = 9), respectively. Late-stage patients, who presented for a second control RT-qPCR, were negative for both assays in most cases. Conversely, neutrophil CD64 expression was unchanged in 75% of cases, without significant differences between groups., Conclusion: Monocyte CD169 evaluation was highly sensitive for detecting SARS-CoV-2 infection in first-presentation patients; and it returns to basal level upon infection clearance. The potential ease of fingerprick collection, minimal time-to-result, and low cost rank this biomarker measurement as a potential viral disease screening tool, including COVID-19. When the virus prevalence in the tested population is usually low (1%-10%), such an approach could increase the testing capacity 10 to 100-fold, with the same limited molecular testing resources, which could focus on confirmation purposes only., Competing Interests: Declaration of conflicting interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: TM, JMB, IAB, PB, and FM are employees of Beckman Coulter Life Sciences, IAB and PB are recipient of a PhD grant from the ANRT (National Agency for Research and Technology). JV has received speaker and consultancy fees from Meda Pharma, Mylan, Novartis, Sanofi, Thermo Fisher, outside this work. The other authors declare no conflict of interest related to this work., (© The Author(s) 2022.)

Background: Sialoadhesin (CD169) has been found to be overexpressed in the blood of COVID-19 patients and identified as a biomarker in early disease. We analyzed CD169 in the blood cells of COVID-19 patients to assess its role as a predictive marker of disease progression and clinical outcomes., Methods: The ratio of the median fluorescence intensity of CD169 between monocytes and lymphocytes (CD169 RMFI) was analyzed by flow cytometry in blood samples of COVID-19 patients (COV) and healthy donors (HDs) and correlated with immunophenotyping, inflammatory markers, cytokine mRNA expression, pulmonary involvement, and disease progression., Results: CD169 RMFI was high in COV but not in HDs, and it correlated with CD8 T-cell senescence and exhaustion markers, as well as with B-cell maturation and differentiation in COV. CD169 RMFI correlated with blood cytokine mRNA levels, inflammatory markers, and pneumonia severity in patients who were untreated at sampling, and was associated with the respiratory outcome throughout hospitalization. Finally, we also report the first evidence of the specific ability of the spike protein of SARS-CoV-2 to trigger CD169 RMFI in a dose-dependent manner in parallel with IL-6 and IL-10 gene transcription in HD PBMCs stimulated in vitro., Conclusion: CD169 is induced by the spike protein and should be considered as an early biomarker for evaluating immune dysfunction and respiratory outcomes in COVID-19 patients.

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., (© 2021 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Introduction: CD64 expression increases on neutrophils during bacterial infections. Recently an increase in CD169 expression has been discovered on monocytes during viral infections. Generally, interferons α (IFNsα) and IFNsγ are key drivers of the infectious host immune response. The purpose of this study was to explore if a link exists between these IFNs and both biomarkers., Methods: Whole blood samples from healthy volunteers were stimulated with either IFNs, interleukins, or infectious extracts, to mimic an infectious state. Expressions of CD64 and CD169 were assessed in these samples by multiple flow cytometry methods, over precise kinetics., Results: The expression of CD64 was statistically higher in samples stimulated with IFNγ, and CD169 in those stimulated with IFNα (and all other type I IFNs). Surface expressions are directly induced by their respective IFNs via Janus kinase/signal transducer and activator of transduction pathways within 6 to 8 hours of incubation. Mixing both types of IFNs seemed to indicate that they partially inhibit each other., Conclusions: The induction of CD169 on monocytes and CD164 on neutrophils by type I and type II IFNs confirms the relevance of these markers for assessing between a viral- vs bacterial-oriented immune response., (© 2020 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)