One-step White Blood Cell Extracellular Staining Method for Flow Cytometry

Belkacem, Inès Ait; Bourgoin, P; Busnel, Jean‐Marc; Galland, Franck; Malergue, Fabrice

doi:10.21769/bioprotoc.4135

Cited by 2 publications

(4 citation statements)

References 11 publications

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“…Three premixed antibody panels were used ( eTable 1 , http://links.lww.com/CCX/B98 ), all obtained from Beckman Coulter (Brea, CA), to assess leucocytes (DURAClone IM Phenotyping Basic dry panel), granulocytes (DURAClone IM Granulocytes dry panel), and myeloid activation markers (IOTest Myeloid Activation Test, liquid cocktail). Leucocyte staining and red blood cell lysis were performed following the one-step method ( 23 ). Further details are available in the Supplemental Digital Content ( http://links.lww.com/CCX/B98 ), and the gating strategy is illustrated in eFigures 1–3 ( http://links.lww.com/CCX/B98 ).…”

Section: Methodsmentioning

confidence: 99%

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The Association of Low CD4 Expression on Monocytes and Low CD8+ T-Cell Count at Hospital Admission Predicts the Need for Mechanical Ventilation in Patients With COVID-19 Pneumonia: A Prospective Monocentric Cohort Study

Allardet-Servent

Belkacem

Miloud³

et al. 2022

Critical Care Explorations

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OBJECTIVES: 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.

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Section: Methodsmentioning

confidence: 99%

“…Recently, a rapid one-step method incorporating whole blood into a standardized no-wash assay has been developed. This provides robust analyses and paves the way for point-of-care application ( 23 , 24 ).…”

mentioning

confidence: 99%

The Association of Low CD4 Expression on Monocytes and Low CD8+ T-Cell Count at Hospital Admission Predicts the Need for Mechanical Ventilation in Patients With COVID-19 Pneumonia: A Prospective Monocentric Cohort Study

Allardet-Servent

Belkacem

Miloud³

et al. 2022

Critical Care Explorations

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“…Donors were not screened for anti-SARS-CoV-2 IgG or IgM levels to assess natural or vaccinal immunization status, but less than 10% individuals had been infected at this time in France 28 . Furthermore, samples were tested double negative for CD169 viral and CD64 bacterial markers and thus patients were considered as infection-free 29 .…”

Section: Samplesmentioning

confidence: 99%

“…Extracellular staining. For immunophenotyping, an infection related panel together with panels dedicated to granulocytes, T cells, B cells and dendritic cells (Table 1) were processed according to the recently developed rapid one-step method 29 . Briefly, whole blood was simultaneously lysed and stained in a lysing/fixative buffer (Versalyse containing Fixative solution 0,05X, Beckman Coulter, Brea, USA) for at least 20 min.…”

Section: Samplesmentioning

confidence: 99%

SARS-CoV-2 spike protein induces a differential monocyte activation that may contribute to age bias in COVID-19 severity

Belkacem

García

Millet-Wallisky³

et al. 2022

Sci Rep

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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.

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One-step White Blood Cell Extracellular Staining Method for Flow Cytometry

Cited by 2 publications

References 11 publications

The Association of Low CD4 Expression on Monocytes and Low CD8+ T-Cell Count at Hospital Admission Predicts the Need for Mechanical Ventilation in Patients With COVID-19 Pneumonia: A Prospective Monocentric Cohort Study

The Association of Low CD4 Expression on Monocytes and Low CD8+ T-Cell Count at Hospital Admission Predicts the Need for Mechanical Ventilation in Patients With COVID-19 Pneumonia: A Prospective Monocentric Cohort Study

SARS-CoV-2 spike protein induces a differential monocyte activation that may contribute to age bias in COVID-19 severity

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