Flow cytometric quantification of neutrophil extracellular traps: Limitations of the methodological approach
Authors: Aneta Manda‐Handzlik, Magdalena Ostafin, Weronika Bystrzycka, Sandra Sieczkowska, Aneta Moskalik, Urszula Demkow, Olga Ciepiela
Journal: American Journal of Hematology (2015)

To the Editor: Neutrophil Extracellular Traps (NETs) are structures composed of DNA, histones, and intracellular enzymes, which are released from granulocytes to immobilize and kill pathogens in blood and tissues 1. Several approaches are used to quantify and assess the effectiveness of NETs’ release, based on fluorescent microscopy, immunoenzymatic measurement of neutrophil elastase (NE) release, and fluorometric measurement of DNA release. Unfortunately, these widely used methods have several drawbacks. Quantification of NETs using fluorescent microscopy is laborious and susceptible to observer bias. On the contrary, the release of NE and DNA is not strictly specific for NETosis as other forms of cell death, such as necrosis, could also result in the elevated levels of extracellular NE and DNA. Thus, it would be very beneficial to develop new and reliable methods to assess NETs’ formation. Recently, Gavillet et al. published an exciting study that describes the flow cytometric method of NETs’ quantification 2. Because we have also been working on the application of flow cytometry for NETs’ studies, we love to share our observations with scientific community. Our major concern regarding the aforementioned work is that Gavillet et al. validated their assay using mice models. But, it is disputable to use murine neutrophils as a reference for human cells—mice release NETs less efficiently than humans, and the formation lasts longer. Thus, it is easier to observe murine NETs using flow cytometry technique because NETs structures are compact and do not show typical morphology of DNA threads 3. It has been reported that only 30% of murine neutrophils release NETs after 16 h of stimulation with 100 nM phorbol-12-myristate-13-acetate (PMA), while approximately 60–80% of human eutrophils release NETs 3 h after stimulation with PMA 3, 4. Gavillet et al. analyzed human samples after 30-min incubation, and the method seemed to be appropriate for such short-time incubation periods. However, our observations suggest that this method cannot be used when long incubation periods are required. Our studies show that human neutrophils stimulated with PMA or calcium ionophore (CI) release NETs very efficiently after 4 h incubation (Fig. 1A). Due to high viscosity of DNA, neutrophils form clumps making it difficult to distinguish single cells. Classical flow cytometry allows to analyze cells in a suspension, and it constitutes the main limitation of flow cytometry-based NETs’ assay. By analyzing human neutrophil samples, we observed a significant loss of cells from suspension after the prolonged incubation. We stimulated 5 × 104 cells in suspension with PMA or CI for 1–3-h incubation and then analyzed the samples with a flow cytometer. Our samples lost neutrophils over time—samples stimulated for 1 h with PMA or CI had ca. 8000 events, while samples stimulated with these compounds for 3 h had only 2500 cells collected during the same time of acquisition (50 s) using flow cytometer (Cytomics FC500, Beckman Coulter). This presents how efficiently human neutrophils release NETs (Fig. 1A). Neutrophils from peripheral blood collected in tubes containing 3.9% sodium citrate were isolated using density gradient and erythrocyte sedimentation in 1% polyvinyl alcohol. Isolated cells (5 × 104 per well) were seeded in LabTek chamber and incubated for 4 h with 100 nM PMA. Then the cells were fixed with 4% paraformaldehyde, stained with 100 nM Sytox green, a fluorescent dye that binds extracellular DNA, and visualized with Leica DMi8 fluorescent microscope. NETs are visible as long clumped threads of DNA released from different granulocytes (Fig. 1A). The number of cells with high expression of citrullinated histones increases over time when neutrophils are incubated with NETs’ inductors. Bars described as 2’, 5’, and 10’ correspond with percentage of citH3+ cells in samples incubated for 2, 5, and 10 min with culture medium, 100 nM PMA and 4 µM CI, respectively. Calculations were performed using Wilcoxon matched-pairs signed rank test (GraphPad Prism 6) and statistically significant difference between analyzed samples is presented in Fig. 1B (p=0.03). Flow cytometric forward scatter/side scatter cytogram showing isolated neutrophils according to the procedure described in Fig. 1A, stimulated for 30 min with 100 nM of PMA. Gating is denoted as follows: S – stimulated cells, N – not affected granulocytes, CD - cell remnants possibly after NETs’ release (Fig. 1C). A cytogram of side scatter/citH3-FITC presents citrullinated histones’ expression from all neutrophils. CitH3 positive events gated in region C (red) fit in CD region of cytogram presented in Fig. 1C, which proves that released NETs are classified in flow cytometer as debris (Fig. 1D). The study was approved by the local ethical committee of Medical University of Warsaw. There are also some doubts regarding “NETs’” gating based on side scatter/forward scatter approaches. In our opinion, the number of cells, which release NETs, could not be properly evaluated. There reason behind this assumption is that many of these cells, as completely disrupted, fall outside the gate (cell remnants) after the formation of NETs (Fig. 1C). Myeloperoxidase (MPO) and citrullinated histones are located outside the cells in fully formed NETs 1, 3, 4. In our opinion, those gated by Gavillet et al., cells are rather stimulated granulocytes with typical for NETosis cellular modifications, which did not release NETs. Neutrophil gating in Fig. 1C shows that cells’ debris contains almost 40% of citrullinated histones, thus we hypothesize that excluding these remnants from the analysis may cause an error in NETs’ flow quantification. We do agree with Gavillet et al. that it is desirable to assess histone citrullination by flow cytometry as a hallmark of NETosis. We have investigated this issue and found that histone citrullination (citH3) increases before tenth minute of neutrophil stimulation—the average amount of neutrophils which expressed citH3 after 2 min was 7.54 ± 4.46% and did not differ between unstimulated, PMA- or CI-treated cells (n=6). There were no changes in the level of citH3 in unstimulated samples over time. The amount of citH3 increased in samples stimulated with PMA (7.25 ± 4.8%, 8.55 ± 5.0%, 10.2 ± 5.8% in 2nd, 5th, and 10th min, respectively) with significant difference between 2nd and 10th min (p=0.03). In CI-stimulated samples, the level of citH3 also increased to 7.7 ± 5.0, 9.03 ± 5.8, 9.03 ± 3.8% over time, but there was no statistical significance (p=0.06 and 0.09 for 2nd vs 5th and 2nd vs 10th min) (Fig. 1B). The level of citrullination after 10th min did not change significantly up to the 3rd h of incubation with NETs’ stimulators. The flow cytometry method for NETs’ quantification is a real challenge as it allows to analyze cells only in suspension. Undoubtedly, Gavillet et al. opened a new field in NETs’ studies. However, the methodology of flow-assisted extracellular traps’ analysis needs significant improvement. Recently, Zhao et al. published a paper based on imaging flow cytometry, which allows for parallel quantification and morphological assessment of NETs. Their analysis is based on the assessment of decondensed nuclei in the cells as a first step in NET formation and analysis of elongation of cells’ shape 5. Nowadays, it seems to be the most suitable NETs’ quantification method for rapid studies in a suspension of cells. Aneta Manda-Handzlik1,2, Magdalena Ostafin1, Weronika Bystrzycka3, Sandra Sieczkowska3, Aneta Moskalik3, Urszula Demkow1, Olga Ciepiela1* 1Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, Warsaw, Poland; 2Postgraduate School of Molecular Medicine, Medical University of Warsaw, Warsaw, Poland; 3Student Scientific Group at Department of Laboratory Diagnostics and Clinical Immunology of Developmental Age, Medical University of Warsaw, Warsaw, Poland Contract grant sponsor: Ministry of Science and Higher Education; Contract grant number: IP2014 006573.