New automated analysis to monitor neutrophil function point-of-care in the intensive care unit after trauma

BACKGROUND: Patients often develop infectious complications after severe trauma. No biomarkers exist that enable early identification of patients who are at risk. Neutrophils are important immune cells that combat these infections by phagocytosis and killing of pathogens. Analysis of neutrophil function used to be laborious and was therefore not applicable in routine diagnostics. Hence, we developed a quick and point-of-care method to assess a critical part of neutrophil function, neutrophil phagosomal acidification. The aim of this study was to investigate whether this method was able to analyze neutrophil functionality in severely injured patients and whether a relation with the development of infectious complications was present. RESULTS: Fifteen severely injured patients (median ISS of 33) were included, of whom 6 developed an infection between day 4 and day 9 after trauma. The injury severity score did not significantly differ between patients who developed an infection and patients who did not (p = 0.529). Patients who developed an infection showed increased acidification immediately after trauma (p = 0.006) and after 3 days (p = 0.026) and a decrease in the days thereafter to levels in the lower normal range. In contrast, patients who did not develop infectious complications showed high-normal acidification within the first days and increased tasset to identify patients at risk for infections after trauma and to monitor the inflammatory state of these trauma patients. CONCLUSION: Neutrophil function can be measured in the ICU setting by rapid point-of-care analysis of phagosomal acidification. This analysis differed between trauma patients who developed infectious complications and trauma patients who did not. Therefore, this assay might prove a valuable asset to identify patients at risk for infections after trauma and to monitor the inflammatory state of these trauma patients. TRIAL REGISTRATION: Central Committee on Research Involving Human Subjects, NL43279.041.13. Registered 14 February 2014. https://www.toetsingonline.nl/to/ccmo_search.nsf/Searchform?OpenForm

The Systemic Immune Response in COVID-19 Is Associated with a Shift to Formyl-Peptide Unresponsive Eosinophils

A malfunction of the innate immune response in COVID-19 is associated with eosinopenia, particularly in more severe cases. This study tested the hypothesis that this eosinopenia is COVID-19 specific and is associated with systemic activation of eosinophils. Blood of 15 healthy controls and 75 adult patients with suspected COVID-19 at the ER were included before PCR testing and analyzed by point-of-care automated flow cytometry (CD10, CD11b, CD16, and CD62L) in the absence or presence of a formyl peptide (fNLF). Forty-five SARS-CoV-2 PCR positive patients were grouped based on disease severity. PCR negative patients with proven bacterial (n = 20) or other viral (n = 10) infections were used as disease controls. Eosinophils were identified with the use of the FlowSOM algorithm. Low blood eosinophil numbers (<100 cells/µL; p < 0.005) were found both in patients with COVID-19 and with other infectious diseases, albeit less pronounced. Two discrete eosinophil populations were identified in healthy controls both before and after activation with fNLF based on the expression of CD11b. Before activation, the CD11b bright population consisted of 5.4% (CI 95% = 3.8, 13.4) of total eosinophils. After activation, this population of CD11b bright cells comprised nearly half the population (42.21%, CI 95% = 35.9, 54.1). Eosinophils in COVID-19 had a similar percentage of CD11b bright cells before activation (7.6%, CI 95% = 4.5, 13.6), but were clearly refractory to activation with fNLF as a much lower percentage of cells end up in the CD11b bright fraction after activation (23.7%, CI 95% = 18.5, 27.6; p < 0.001). Low eosinophil numbers in COVID-19 are associated with refractoriness in responsiveness to fNLF. This might be caused by migration of fully functional cells to the tissue

An increase in CD62Ldim neutrophils precedes the development of pulmonary embolisms in COVID-19 patients

OBJECTIVES: A high incidence of pulmonary embolism (PE) is reported in patients with critical Corona Virus Disease 2019 (COVID-19). Neutrophils may contribute to this through a process referred to as immunothrombosis. The aim of this study was to investigate the occurrence of neutrophil subpopulations in blood preceding the development of COVID-19 associated PE. METHODS: We studied COVID-19 patients admitted to the ICU of our tertiary hospital between 19-03-2020 and 17-05-2020. Point-of-care fully automated flow cytometry was performed prior to ICU admission, measuring the neutrophil activation/maturation markers CD10, CD11b, CD16 and CD62L. Neutrophil receptor expression was compared between patients who did or did not develop PE (as diagnosed on CT angiography) during or after their ICU stay. RESULTS: Among 25 eligible ICU patients, 22 subjects were included for analysis, of whom nine developed PE. The median (IQR) time between neutrophil phenotyping and PE occurrence was 9 (7-12) days. A significant increase in the immune-suppressive neutrophil phenotype CD16bright /CD62Ldim was observed on the day of ICU admission (p=0.014) in patients developing PE compared to patients who did not. CONCLUSION: The increase in this neutrophil phenotype indicates that the increased number of CD16bright /CD62Ldim neutrophils might be used as prognostic marker to predict those patients that will develop PE in critical COVID19 patients

Kinetics of Neutrophil Subsets in Acute, Subacute, and Chronic Inflammation

At homeostasis the vast majority of neutrophils in the circulation expresses CD16 and CD62L within a narrow expression range, but this quickly changes in disease. Little is known regarding the changes in kinetics of neutrophils phenotypes in inflammatory conditions. During acute inflammation more heterogeneity was found, characterized by an increase in CD16dim banded neutrophils. These cells were probably released from the bone marrow (left shift). Acute inflammation induced by human experimental endotoxemia (LPS model) was additionally accompanied by an immediate increase in a CD62Llow neutrophil population, which was not as explicit after injury/trauma induced acute inflammation. The situation in sub-acute inflammation was more complex. CD62Llow neutrophils appeared in the peripheral blood several days (>3 days) after trauma with a peak after 10 days. A similar situation was found in the blood of COVID-19 patients returning from the ICU. Sorted CD16low and CD62Llow subsets from trauma and COVID-19 patients displayed the same nuclear characteristics as found after experimental endotoxemia. In diseases associated with chronic inflammation (stable COPD and treatment naive HIV) no increases in CD16low or CD62Llow neutrophils were found in the peripheral blood. All neutrophil subsets were present in the bone marrow during homeostasis. After LPS rechallenge, these subsets failed to appear in the circulation, but continued to be present in the bone marrow, suggesting the absence of recruitment signals. Because the subsets were reported to have different functionalities, these results on the kinetics of neutrophil subsets in a range of inflammatory conditions contribute to our understanding on the role of neutrophils in health and disease

Flow cytometric evaluation of the neutrophil compartment in COVID-19 at hospital presentation: A normal response to an abnormal situation

Coronavirus disease 2019 (COVID-19) is a rapidly emerging pandemic disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Critical COVID-19 is thought to be associated with a hyper-inflammatory process that can develop into acute respiratory distress syndrome, a critical disease normally mediated by dysfunctional neutrophils. This study tested the hypothesis whether the neutrophil compartment displays characteristics of hyperinflammation in COVID-19 patients. Therefore, a prospective study was performed on all patients with suspected COVID-19 presenting at the emergency room of a large academic hospital. Blood drawn within 2 d after hospital presentation was analyzed by point-of-care automated flow cytometry and compared with blood samples collected at later time points. COVID-19 patients did not exhibit neutrophilia or eosinopenia. Unexpectedly neutrophil activation markers (CD11b, CD16, CD10, and CD62L) did not differ between COVID-19-positive patients and COVID-19-negative patients diagnosed with other bacterial/viral infections, or between COVID-19 severity groups. In all patients, a decrease was found in the neutrophil maturation markers indicating an inflammation-induced left shift of the neutrophil compartment. In COVID-19 this was associated with disease severity

Intracellular Penetration and Effects of Antibiotics on Staphylococcus aureus Inside Human Neutrophils : A Comprehensive Review

Neutrophils are important assets in defense against invading bacteria like staphylococci. However, (dysfunctioning) neutrophils can also serve as reservoir for pathogens that are able to survive inside the cellular environment. Staphylococcus aureus is a notorious facultative intracellular pathogen. Most vulnerable for neutrophil dysfunction and intracellular infection are immune-deficient patients or, as has recently been described, severely injured patients. These dysfunctional neutrophils can become hide-out spots or "Trojan horses" for S. aureus. This location offers protection to bacteria from most antibiotics and allows transportation of bacteria throughout the body inside moving neutrophils. When neutrophils die, these bacteria are released at different locations. In this review, we therefore focus on the capacity of several groups of antibiotics to enter human neutrophils, kill intracellular S. aureus and affect neutrophil function. We provide an overview of intracellular capacity of available antibiotics to aid in clinical decision making. In conclusion, quinolones, rifamycins and sulfamethoxazole-trimethoprim seem very effective against intracellular S. aureus in human neutrophils. Oxazolidinones, macrolides and lincosamides also exert intracellular antibiotic activity. Despite that the reviewed data are predominantly of in vitro origin, these findings should be taken into account when intracellular infection is suspected, as can be the case in severely injured patients

Kinetics of Neutrophil Subsets in Acute, Subacute, and Chronic Inflammation

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The Systemic Immune Response in COVID-19 Is Associated with a Shift to Formyl-Peptide Unresponsive Eosinophils

A malfunction of the innate immune response in COVID-19 is associated with eosinopenia, particularly in more severe cases. This study tested the hypothesis that this eosinopenia is COVID-19 specific and is associated with systemic activation of eosinophils. Blood of 15 healthy controls and 75 adult patients with suspected COVID-19 at the ER were included before PCR testing and analyzed by point-of-care automated flow cytometry (CD10, CD11b, CD16, and CD62L) in the absence or presence of a formyl peptide (fNLF). Forty-five SARS-CoV-2 PCR positive patients were grouped based on disease severity. PCR negative patients with proven bacterial (n = 20) or other viral (n = 10) infections were used as disease controls. Eosinophils were identified with the use of the FlowSOM algorithm. Low blood eosinophil numbers (&lt;100 cells/μL; p &lt; 0.005) were found both in patients with COVID-19 and with other infectious diseases, albeit less pronounced. Two discrete eosinophil populations were identified in healthy controls both before and after activation with fNLF based on the expression of CD11b. Before activation, the CD11bbright population consisted of 5.4% (CI95% = 3.8, 13.4) of total eosinophils. After activation, this population of CD11bbright cells comprised nearly half the population (42.21%, CI95% = 35.9, 54.1). Eosinophils in COVID-19 had a similar percentage of CD11bbright cells before activation (7.6%, CI95% = 4.5, 13.6), but were clearly refractory to activation with fNLF as a much lower percentage of cells end up in the CD11bbright fraction after activation (23.7%, CI95% = 18.5, 27.6; p &lt; 0.001). Low eosinophil numbers in COVID-19 are associated with refractoriness in responsiveness to fNLF. This might be caused by migration of fully functional cells to the tissue