Abstract
This thesis describes the introduction of a new method for bedside analysis of innate immune cells in health and disease. Flow cytometry is an analysis method, that is almost 50 years old and has been extensively used in biomedical research to investigate cellular responses as described in chapter 2. Recently,
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fully automated flow cytometry was developed, which resulted in huge advantages and improvements in the analysis of innate immune cells. Chapter 3 of this thesis highlights the following improvements in comparison with conventional flow cytometry:
1. Fully automated flow cytometry reduces analysis time from 2 h to only 20 min; this results in more accurate knowledge of ‘near-real’ in-vivo cell biology.
2. Fully automated flow cytometry results in less in-vitro manipulation steps, enabling better reproducibility.
3. Fully automated flow cytometry requires no dedicated lab personnel, enabling point-of-care application(s).
This new method has been used to investigate ‘near-real’ in-vivo cell biology in chapters 4, 5 and 6. These studies resulted in the following conclusions.
This new method is used to investigate innate immune cells in COVID-19 patients (chapters 4 and 5), resulting in the following conclusions. COVID-19 severity is associated with a maturation dissociation in the complete neutrophil compartment characterized by an overall low FcγIII/CD16 and Neprilysin/CD10 expression (chapter 4). The lack of expression of Neprilysin/CD10 might contribute to the compromised bradykinin pathway in COVID-19 patients (chapter 4). No signs of hyper inflammation nor activation in the neutrophils in the peripheral blood at hospital admission is found (chapter 4). An increase of CD16bright/CD62Ldim neutrophils is found prior to intensive care unit (ICU) admission in critically ill COVID-19 patients developing pulmonary embolisms compare to ICU patients who did not develop pulmonary embolisms (chapter 5). This may provide the missing link between altered hemostasis and malfunction of the immune system in the pathogenesis of pulmonary embolisms in critical COVID-19 patients.
Fully automated flow cytometry in a field laboratory is used to analyze patient with inflammatory bowel disease (ulcerative colitis and Crohn's disease). This study shows that decreased responsiveness of neutrophils and monocytes to fMLF was demonstrated after repetitive bouts of prolonged exercise in these patients (chapter 6). These refractory cells might create a lower inflammatory state in the intestine, providing a putative mechanism for the decrease in flare-ups in these patients after repeated exercise.
Fully automated flow cytometry also enabled clinical applicability in daily patient care. Chapters 7, 8 and 9 not only investigated the ‘near-real’ in-vivo cell biology but also provided clinical correlations with specific cell markers.
Point-of-care fully automated flow cytometry is was used to study neutrophil responses in trauma patients in chapters 8 and 9. Implementation of point-of-care fully automated flow cytometry in the trauma room appeared feasible (chapter 9). Neutrophil phagosomal acidification differs between patients who develop infectious complications and patients who do not (chapter 8). The assessment of CD16dim/CD62Lbright neutrophils is used for early detection of patients at risk for infectious complications (AUC = 0.90) (chapter 9). The %CD16dim/CD62Lbright neutrophils provided valuable information for clinical decision marking in trauma patients (chapter 9). With the results of chapter 9, the trauma surgery department of the UMC Utrecht has decided to implement this analysis as a standard-of-care procedure for support in clinical decision marking. To further elaborate the results of chapter 9 in a multivariate model, an international multicenter study is initiated. Chapter 10 shows a study protocol for the development and testing of a multivariate prediction model in a multicenter study.
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