Differential presence of neutrophil extracellular traps in serum vs plasma of patients with critical limb-threatening ischemia.

BACKGROUND

Neutrophil extracellular traps (NETs) contribute to both arterial and venous thrombosis. However, their detection in peripheral blood remains challenging, with circulating DNA and nucleosomes often used as surrogate markers.

OBJECTIVES

This study evaluated circulating NETs, nucleosomes, and double-stranded DNA (dsDNA) in different blood matrices, serum vs plasma, for the quantification of NET markers.

METHODS

Thirteen patients with end-stage critical limb-threatening ischemia (CLTI) and 7 controls were included in this retrospective study. NETs (myeloperoxidase [MPO]-DNA complexes), nucleosomes, and dsDNA were quantified in serum and plasma samples from patients with CLTI, and in serum and various plasma types (EDTA, citrate, and heparin) in controls.

RESULTS

MPO-DNA complexes were undetectable in plasma but significantly elevated in the serum of patients with CLTI compared with controls (median optical density, 0.048 vs 0.004; P = .012). Serum levels of nucleosomes and dsDNA were also significantly higher in patients with CLTI than in controls (P = .029 and P = .019, respectively) and higher in serum than in plasma (P < .01). In controls, dsDNA and nucleosomes were also higher in serum than in plasma, but MPO-DNA complexes remained undetectable. Correlations among MPO-DNA, nucleosomes, and dsDNA were observed only in serum.

CONCLUSION

NETs, as evidenced by MPO-DNA complexes, were detectable only in the serum of patients with CLTI, supporting a disease-related origin. Our findings suggest that NET markers are more readily detectable in serum. However, the potential influence of coagulation-related processes during serum preparation should be considered. Our results highlighted the need for methodological standardization in NET biomarker measurements in clinical studies.