PB0785 - NETosis and DNAses exploration Development of an assay to explore total DNAse activity and pre-analytical considerations for NETosis markers et DNAses activity.

Background: Neutrophil Extracellular Traps (NETs) are web-like structures released by neutrophils to fight pathogens. They are degraded by DNases. Plasmatic NETs markers can be measured by ELISA. A work by an ISTH subcommittee to standardize NETs quantification is ongoing, but pre-analytical conditions for reliable measure of NETs markers need to be define. Moreover, available techniques to explore assess DNAse activity lack reproducibility and repeatability and make them not suitable for the study of large cohorts.

Aims: To develop a new assay to quantify total DNAse activity in human samples and define pre-analytical conditions for reliable assessment of NETs markers and DNAse in patients.

Methods: We studied 3 validated NETs markers: MPO-DNA complex, citrullinated histone H3 (H3cit) and H3cit-DNA complex. We determined the influence of the type of anticoagulant, thawing-frozing cycles and Hemolysis-Icterus-Lipemia (HIL) on NETs markers levels assessment. We also determined NETs markers’ stability during time after blood sampling. Plasmatic DNAse activity was determined by the quantification of residual DNA by fluorimetry using PicoGreen® reagent. Finally we defined the pre-analytical conditions for reliable measurement of DNAse activity.

Results: Citrated plasma was the only anticoagulant allowing reproducible assessment of the 3 NETs markers. For reliable measures, the maximum delay between blood sampling and plasma thawing was 24h, 6h and 2h for MPO-DNA, H3Cit and H3Cit-DNA respectively. Total DNase activity was assessed on citrated plasma with good intra-assay and inter-assay reproducibility (coefficient of variation 6,7 to 10% and 7,8 to 16,5% respectively) and can be realized up to 24 hours after blood sampling. HIL+ and thawing-frozing cycles disturbed both NETs markers and DNAse activity measurement.

Conclusion(s): We developped a new and reproducible method to evaluate DNAse activity on human samples and defined pre-analytical conditions to reliably measure NETs markers and DNase activity.