Response to correspondence: Flow cytometric quantification of neutrophil extracellular traps: Limitations of the methodological approach by Ciepiela et al.
Authors: David A. Williams, Mathilde Gavillet
Journal: American Journal of Hematology (2016)

Neutrophil extracellular traps (NETs) are released by neutrophils upon certain stress stimuli and are believed to contribute to physiological processes, such as innate immunity, as well as numerous diseases (deep vein thrombosis, myocardial ischemia, autoimmune diseases, and others). We recently published in the American Journal of Hematology 1 a new methodological approach for NETs quantification based on flow cytometry. We have been pleased to see this publication has received much attention from our colleagues. Among those, Ciepiela et al. 2 submitted a letter containing their unpublished data and discussed the limitations of the currently available NETs assays, including ours. However, we respectfully disagree with several of their assertions. First, acknowledging the structural differences between murine neutrophil-derived NETs and human neutrophil-derived NETS, we independently validated our method on both type of samples (Figs. 1 and 2, respectively). Furthermore, the existence of NETs was first identified in an animal model by Brinkmann et al. 3 and since then, numerous published data have established the presence of murine NETosis using both in vitro assays and its relevance in murine models in vivo 4-6 With respect to the comment by Ciepiela et al. about shorter incubation times, these are indeed more suited for experiments involving human neutrophils and strong NETs inductors such as ionomycin. We agree with these authors that differing incubation times may well lead to different results. However, this in no way invalidates our findings. Indeed, NETs tend to degrade and/or form clumps after prolonged incubation in vitro. We thus would recommend using the experimental conditions described in our publication, keep handling times to a minimum, and keeping the volume sufficient to avoid formation of larger aggregates. Providing these conditions are fulfilled, we observed only minimal doublet formation and no large aggregates allowing adequate quantification by flow cytometry. We agree that flow analysis for NETs using our described method is best not gated based on forward and side scatter properties, which are very heterogeneous for NETs in suspension. Indeed this gating step is probably dispensable. As described in the methods section and in the supplementary figures of our paper, NETs are efficiently identified as H3Cit positive, MPO positive, and DAPI positive. The fact that histone citrullinization (H3Cit) is a necessary and early step of NETosis is a well-established notion, supported by a large body of published experimental data 6-8. Thus, we would suggest the data presented by Ciepiela et al. are inconsistent with other published data. Indeed, 4 µM Ionomycin is a strong inducer of H3Cit and its effect is observable well beyond 10 min 6. While we agree that NETs quantification by flow can be further optimized, we disagree with the interpretation by Ciepiela et al. regarding the appropriateness of one method of NETs determination versus the other. In fact, we believe the arguments they have provided do not justify their conclusion on this point. As stated in our paper, the flow-based method, based on immunological detection of NETs components, circumvents two main limitations of the microscopic assay in that it is observer-independent and allows robust and rapid assessment of a much larger number of cells per sample. Furthermore, we feel that our method is ideally designed for future clinical use because it allows for direct assessment of NETs formed in vivo from peripheral blood samples. This is in contrast to microscopy-based analysis. Although elegant and allowing refined analysis, the image-based quantitative method recently described by Zhao et al. 9 requires high technical expertise that may be accessible in only a few facilities, without obvious improvement in the robustness of the flow cytometric quantification described in our publication. In addition, our method enables fluorescence activated cell sorting of NETs in clinical settings. The simplicity of the method we describe and the use of nearly universally available equipment may thus facilitate new avenues of clinical and biological investigations. Our hope is that our work, alongside others, will continue to foster discussion and allow increasing the understanding of NETs biology and its potential clinical applications. David A. Williams1,2,3*, Mathilde Gavillet1 1Division of Hematology/Oncology, Boston Children's Hospital, Boston, Massachusetts; 2Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts; 3Department of Pediatrics, Harvard Medical School, Boston, Massachusetts