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Flow cytometric quantification of neutrophil extracellular traps: Limitations of the methodological approach 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 CORRESPONDENCE the average amount of neutrophils which expressed citH3 after 2 min was 7.54 6 4.46% and did not differ between unstimulated, PMA-or CI-treated cells (n56). 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 6 4.8%, 8.55 6 5.0%, 10.2 6 5.8% in 2nd, 5th, and 10th min, respectively) with significant difference between 2nd and 10th min (p50.03). In CI-stimulated samples, the level of citH3 also increased to 7.7 6 5.0, 9.03 6 5.8, 9.03 6 3.8% over time, but there was no statistical significance (p50.06 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 ...

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Acute leukemias, the most common cancers in children, are characterized by excessive proliferation of malignant progenitor cells. As a consequence of impaired blood cell production, leukemia patients are susceptible to infectious complications—a major cause of non-relapse mortality. Neutrophil extracellular traps (NETs) are involved in various pathologies, from autoimmunity to cancer. Although aberrant NETs formation may be partially responsible for immune defects observed in acute leukemia, still little is known on the NET release in the course of leukemia. Here, we present the first comprehensive evaluation of NETs formation by neutrophils isolated from children with acute leukemia in different stages of the disease and treatment stimulated in vitro with phorbol 12-myristate 13-acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore (CI). NETs release was measured using quantitative fluorescent method and visualized microscopically. In this setting, NETs release was significantly impaired in leukemic children both at the diagnosis and during the treatment, and full restoration of neutrophil function was achieved only after successful completion of the leukemia treatment. We suggest that neutrophil function impairment may result from both disease- and treatment-related factors. In this context, deficient innate immune response observed in acute leukemia patients may be present regardless of neutrophil count and contribute to secondary immunodeficiency observed in this population.