Flow Cytometry-Based Quantification of Neutrophil Extracellular Traps Shows an Association with Hypercoagulation in Septic Shock and Hypocoagulation in Postsurgical Systemic Inflammation-A Proof-of-Concept Study

Abstract

This proof-of-concept study aimed to evaluate a novel method of flow cytometry-based quantification of neutrophil extracellular traps (NETs) in septic shock patients and to identify possible interactions between the number of free-circulating NETs and alterations of the coagulatory system. Patients suffering from septic shock, a matched control group (CTRL), and patients suffering from systemic inflammation after cardiac (CABG) or major abdominal surgery (MAS) were enrolled in this prospective proof-of-concept study. Compared to the matched controls, free-circulating NETs were significantly elevated in septic shock and postsurgical patients (data are presented in median (IQR)); septic shock: (2.7 (1.9-3.9); CABG: 2.7 (2.1-3.7); MAS: 2.7 (2.1-3.9); CTRL: 1.6 (1-2); CTRL vs. septic shock: p = 0.001; CTRL vs. CABG: p < 0.001; CTRL vs. MAS: p < 0.001). NETs correlated positively with FIBTEM mean clot firmness (MCF) in septic shock patients (r = 0.37, p < 0.01) while they correlated negatively in surgical patients (CABG: r = -0.28, p < 0.01; MAS: r = -0.25, p = 0.03). Flow-cytometric quantification of NETs showed a significant increase in free-circulating NETs under inflammatory conditions. Furthermore, this study hints to an association of the number of NETs with hypercoagulation in septic shock patients and hypocoagulation in surgery-induced inflammation.

Keywords: cardiopulmonary bypass; coagulopathy; inflammation; major abdominal surgery; sepsis.

Figure 1

Description of the flow-cytometric gating strategy. First, leucocytes were targeted (A) and neutrophils identified as CD15⁺-cells (B). Second, the fluorescence-minus-one (FMO) technique was used to set the gating borders of MPO- and anti-H3-Histone-antibody (C,D) and last, MPO- and anti-H3-Histone-positive cells were defined as surrogates for NETs (E). Results are shown as the percentage of gated neutrophils (F, red box). Abbreviations: APC: Allophycocyanin; CD: Cluster of Differentiation; FITC: Fluorescein isothiocyanate; FSC: Forward Scatter; MPO: Myeloperoxidase; NETs: Neutrophil Extracellular Traps; SSC: Side Scatter.

Figure 2

Results of the NET quantification of the study groups. With the exception of preoperative values, all time points per group were summarized. Results are shown in boxplot diagrams. Asterisks display the degree of statistical significance: **: p < 0.01; ***: p < 0.001. Abbreviations: NETs: Neutrophil Extracellular Traps.

Figure 3

Time courses of free-circulating NETs. Results are shown in boxplot diagrams. Asterisks display the degree of statistical significance: *: p < 0.05; **: p < 0.01; ***: p < 0.001. Abbreviations: CTRL: Control group; NETs: Neutrophil Extracellular Traps.

Figure 4

Positive correlation of free-circulating NETs to FIBTEM MCF in septic shock patients. Time courses of NETs (A,D), FIBTEM MCF (B), and EXTEM MCF (E) are shown as boxplot diagrams while scatter plots are uses to present correlations between NETs and FIBTEM MCF (C) and EXTEM MCF (F). Abbreviations: CTRL: Control group; MCF: Mean Clot Firmness; NETs: Neutrophil Extracellular Traps; r: Pearson’s Correlation Coefficient.

Figure 5

Inverse correlation of free-circulating NETs to FIBTEM MCF in cardiac surgical patients. Time courses of NETs (A,D), FIBTEM MCF (B), and EXTEM MCF (E) are shown as boxplot diagrams while scatter plots are used to present correlations between NETs and FIBTEM MCF (C) and EXTEM MCF (F). Abbreviations: CTRL: Control group; MCF: Mean Clot Firmness; NETs: Neutrophil Extracellular Traps; r: Pearson’s Correlation Coefficient.

Figure 6

Inverse correlation of free-circulating NETs to FIBTEM MCF in major abdominal surgical patients. Time courses of NETs (A,D), FIBTEM MCF (B), and EXTEM MCF (E) are shown as boxplot diagrams while scatter plots are uses to present correlation between NETs and FIBTEM MCF (C) and EXTEM MCF (F). Abbreviations: CTRL: Control group; MCF: Mean Clot Firmness; NETs: Neutrophil Extracellular Traps; r: Pearson’s Correlation Coefficient.