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Table 2 Criteria for assessment of neutrophils and NETs in categories

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

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
  1. 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