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Background: Innate effector cells are very responsive to infectious and inflammatory cues found in damaged and inflamed tissues. Their activation is a potential target to assess the state of the immune system. Unfortunately, these cells are very susceptible for ex-vivo activation, hampering accurate interpretation of flow cytometry data. Whether a brief window exists before ex-vivo activation starts to occur is currently unknown. Aims: 1) This study extensively investigated ex-vivo activation of innate effector cells over time. 2) We tested the feasibility of applying a mobile, automated, flow cytometry laboratory for out-of-hospital Point-of-Care analyses to minimize ex-vivo activation bias. Methods: 1) Ex-vivo neutrophil, eosinophil and monocyte activation in a blood collection tube over time and the reactivity to a formyl-peptide was investigated in a healthy cohort. 2) To facilitate fast, out-of-hospital analysis, application of the mobile flow cytometry was tested by placing an automated flow cytometer into a van. The stability of the setup was assessed by repetitively measuring laser alignment and fluorescence verification beads. Findings: 1) Immediately after venipuncture activation marker expression on neutrophils, eosinophils and monocyte subsets started to change in a time-dependent manner. 2) The mobile flow cytometry laboratory travelled over 3000 km, performing measurements at 19 locations with a median single-person-set-up time of 14 min. The laser alignment and fluorescence were stable during all experiments. Conclusions: Accurate flow data of innate immune cells are only obtained when ex-vivo activation is kept to minimum. The use of a mobile, fast, automated, flow cytometry laboratory for out-of-hospital Point-of-Care analyses provides new investigational and diagnostic possibilities outside major hospital flow cytometry laboratories.
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Introduction: Neutrophil and eosinophil activation and its relation to disease severity has been understudied in primary care patients with COVID-19. In this study, we investigated whether the neutrophil and eosinophil compartment were affected in primary care patients with COVID-19. Methods: COVID-19 patients, aged ≥ 40 years with cardiovascular comorbidity presenting to the general practitioner with substantial symptoms, partaking in the COVIDSat@Home study between January and April 2021, were included. Blood was drawn during and 3 to 6 months after active COVID-19 disease and analyzed by automated flow cytometry, before and after stimulation with a formyl-peptide (fNLF). Mature neutrophil and eosinophil markers at both time points were compared to healthy controls. A questionnaire was conducted on disease symptoms during and 3 to 6 months after COVID-19 disease. Results: The blood of 18 COVID-19 patients and 34 healthy controls was analyzed. During active COVID-19 disease, neutrophils showed reduced CD10 (p = 0.0360), increased CD11b (p = 0.0002) and decreased CD62L expression (p < 0.0001) compared to healthy controls. During active COVID-19 disease, fNLF stimulated neutrophils showed decreased CD10 levels (p < 0.0001). Three to six months after COVID-19 disease, unstimulated neutrophils showed lowered CD62L expression (p = 0.0003) and stimulated neutrophils had decreased CD10 expression (p = 0.0483) compared to healthy controls. Both (un)stimulated CD10 levels increased 3 to 6 months after active disease (p = 0.0120 and p < 0.0001, respectively) compared to during active disease. Eosinophil blood counts were reduced during active COVID-19 disease and increased 3 to 6 months after infection (p < 0.0001). During active COVID-19, eosinophils showed increased unstimulated CD11b (p = 0.0139) and decreased (un)stimulated CD62L expression (p = 0.0036 and p = 0.0156, respectively) compared to healthy controls. Three to six months after COVID-19 disease, (un)stimulated eosinophil CD62L expression was decreased (p = 0.0148 and p = 0.0063, respectively) and the percentage of CD11bbright cells was increased (p = 0.0083 and p = 0.0307, respectively) compared to healthy controls. Conclusion: Automated flow cytometry analysis reveals specific mature neutrophil and eosinophil activation patterns in primary care patients with COVID-19 disease, during and 3 to 6 months after active disease. This suggests that the neutrophil and eosinophil compartment are long-term affected by COVID-19 in primary care patients. This indicates that these compartments may be involved in the pathogenesis of long COVID.