Neutrophil extracellular trap (NET) formation characterises stable and exacerbated COPD and correlates with airflow limitation

Table 2 Criteria for assessment of neutrophils and NETs in categories

Category	Predefined criteria
Non-activated neutrophil	intact cell	lobulated nucleus	positive NE staining in cytoplasmic granulae only
Activated/NET-forming neutrophil	intact cell	altered nuclear morphology	positive NE staining in cytoplasm and/or nucleus
Large amounts of NETs	extracellular fibrous structures	positive PI-staining	positive NE staining	extended, confluent and/or overlapping formations occupying at least 1 mm²
Minor traces of NETs	extracellular fibrous structures	positive PI-staining	positive NE staining	≤10 small-sized (≤50 μm²) nonoverlapping items per 100 mm²

Classification into a category requires fulfillment of all criteria listed for that category. Note that an individual sputum sample (and all CLSM specimens prepared from it) could apply to both neutrophil categories (‘non-activated’ and ‘activated/NET-forming’). By contrast, an individual sputum sample could not apply to both NET-related categories because a sample was assigned to the ‘large amounts’ category when only one CLSM specimen fulfilled the criteria for this category, irrespective of whether any other specimen from the same sample fulfilled only the criteria for ‘minor traces’, or was free of NETs

ISSN: 1465-993X