The standardization of NET measurements, a work in progress.

Posted on: October 5, 2023

-Contributed by Abi Kasberg, PhD and Olivia Stricker, PhD

 

Neutrophil extracellular traps (NETs) are an amazing participant of the innate immune system. In a first line of defense, neutrophils expel decondensated chromatin into the extracellular matrix to trap invading pathogens in a mechanism called NETosis. NETs are made up of DNA, histones, and granule proteins which have antimicrobial properties. When dysregulated, NET components can have procoagulant, proinflammatory, and toxic effects. Studying NETs and the process of NETosis is a relatively new and fascinating area of research for thrombosis, haemostasis, autoimmunity, and cancer studies.

 

Problems

There are several confounding hurdles surrounding the research of NETs and NETosis. Due to a lack of available and accurate commercial assays, in-house protocols have been developed to evaluate NETosis. The use of non-standardized tools to measure NETs is problematic for the following reasons:

  1. Lack of standardization in assay methodologies and techniques drive variability in results.
  2. There is insufficient continuity to be able to accurately compare NETosis readouts between studies or across diseases.
  3. The field is unable to accurately distinguish between the types of NETs, the identification of extracellular traps (ETs) that originate from non-neutrophils, or account for distinct signaling pathways that drive NETosis.

 

 

NETs Standardization Project

To get ahead of this, an initiative has been created to define and standardize methodologies surrounding the measurement of NETs. The ISTH SSC subcommittee on Vascular Biology is systematically and thoughtfully assessing the tools that are being used to measure NETs, the consistency of assay outputs, and the mechanistic value that each brings to the bench. The goal is to develop a “Net Profile” to measure NETs in a standardized and consistent way. Roughly three project phases have been initiated to pursue this collaborative project:

Phase 1: Methodology – Survey researchers who are currently measuring NETs and gather information about the methodologies that are being used.

Phase 2: Samples – Send prepared, characterized plasma samples to individual research laboratories to measure NETs using their own developed methodologies.

Phase 3: Based on the outcomes of phase 2, send prepared, characterized plasma samples to individual research labs to use in designated protocols, including a commercially-available assay from Volition Therapeutics that measures nucleosomes1.

 

 

Incoming Results

Preliminary study data has been presented orally at ISTH meetings with anticipated publications to come.

Phase 1 results showed that out of 20 labs measuring MPO+DNA, there were no labs that used the same sample preparation or assay protocol. This revealed that there is a dramatic lack of standardization in the tools and assays used to measure NETs. This is particularly important for sample preparation and experiment setup, because different neutrophil stimulation protocols can yield different outcomes.

Phase 2 results showed that the prepared samples that were sent to each lab for NET characterization were giving different results. This suggests that the NET measurement assays are not all measuring the same thing.

Phase 3 is in progress.

What is apparent from the results reported from phases 1 and 2, is that there is a clear lack of standardization in the current protocols and sample preparations being used to measure NETs. This led to the subcommittee developing assay recommendations in 2022 that cover the mechanistic span of NETosis. To best capture the complete scope of NETosis, measurements should be acquired from each of the following three categories:

  • Cell free DNA (cfDNA)
  • Citrullinated H3 histones (citH3)
  • Granule protein-DNA complexes

These recommendations emphasize the need for commercially-available assays in each category that can be used to accelerate the standardization of NET measurements in research.

 

 

Nomenclature Concerns

In addition to a lack of standardization in research methodologies, the consistent dissemination of research findings within and across research interests is hampered by differences in nomenclature use. Several terms have appeared in published literature regarding types of NETosis. These different terms are frequently used interchangeably, which can muddy the interpretation of research outcomes.

The term “NETosis” is inherently assumed to be specific to neutrophils. However, neutrophils are not the only cell type to release extracellular traps. Other immune cells including eosinophils, basophils, and even cancer cells can expel extracellular traps. Thus, it is not always obvious or known which cell types are being specified in the chosen nomenclature.

Efforts need to be made to identify and distribute the best practices for NETosis nomenclature use. Currently, it is not always obvious what the differences are between terms. Here are some breakdowns of commonly used NETosis terms that we have run across in the literature.

Suicidal or lytic NETosis describes neutrophil death as the result of NET expulsion from the cell membrane. This can include DNA from nuclear or mitochondrial origin.

Survival or vital NETosis describes when the neutrophil survives following NETosis. This can occur by the gentle release of chromosomal DNA from the cell via secretory vesicles. The active release of mitochondrial DNA (mtDNA) without nuclear DNA (nDNA) can also result in vital NETosis.

Mitochondrial DNA extracellular traps describes the release of mtDNA instead of nDNA during NETosis. Neutrophil release of mtDNA results in cell viability.

 

Additional work is being invested to better understand the priming conditions, physical triggers, and signaling pathways that drive the release of each type of NET. This research will provide valuable insight to not only accelerate NET measurement standardizations, it will also aid in clarifying the differences between known neutrophil functions; phagocytosis, degranulation, suicidal NETosis, and survival NETosis.

 

 

 

  1. The inclusion of the Volition Therapeutics nucleosome kit is not an endorsement from the ISTH SSC Subcommittee on Vascular Biology or the NETs Standardization Project.

 

 

 

 

Resources

Vascular Biology Subcommittee. Towards standardization of neutrophil extracellular trap (NET) measurements in patient samples. Accessed on September 15, 2023 https://cdn.ymaws.com/www.isth.org/resource/resmgr/ssc/ssc_subcommittee_project_mar.pdf