Markers of neutrophil activation and neutrophil extracellular traps in diagnosing patients with acute venous thromboembolism: A feasibility study based on two VTE cohorts

Abstract

Background Venous thromboembolism (VTE) diagnosis would greatly benefit from the identification of novel biomarkers to complement D-dimer, a marker limited by low specificity. Neutrophil extracellular traps (NETs) have been shown to promote thrombosis and could hypothetically be used for diagnosis of acute VTE. Objectives To assess the levels of specific markers of neutrophil activation and NETs and compare their diagnostic accuracy to D-dimer. Methods We measured plasma levels of neutrophil activation marker neutrophil elastase (NE), the NET marker nucleosomal citrullinated histone H3 (H3Cit-DNA) and cell-free DNA in patients (n = 294) with suspected VTE (pulmonary embolism and deep vein thrombosis) as well as healthy controls (n = 30). A total of 112 VTE positive and 182 VTE negative patients from two prospective cohort studies were included. Results Higher levels of H3Cit-DNA and NE, but not cell-free DNA, were associated with VTE. Area under receiver operating curves (AUC) were 0.90 and 0.93 for D-dimer, 0.65 and 0.68 for NE and 0.60 and 0.67 for H3Cit-DNA in the respective cohorts. Adding NE and H3Cit-DNA to a D-dimer based risk model did not improve AUC. Conclusions Our study demonstrates the presence of neutrophil activation and NET formation in VTE using specific markers. However, the addition of NE or H3Cit-DNA to D-dimer did not improve the discrimination compared to D-dimer alone. This study provides information on the feasibility of using markers of NETs as diagnostic tools in acute VTE. Based on our findings, we believe the potential of these markers are limited in this setting.

Biomarkers of cell damage, neutrophil and macrophage activation associated with in-hospital mortality in geriatric COVID-19 patients

“…In addition, the elevated plasma levels of NET markers found in COVID-19 patients when compared to uninfected controls in the above cited study [ 34 ], could be considered in overall agreement with our results of an increased abundance of n-cfDNA (Alu 115 and Alu 247 DNA) in infected versus uninfected geriatric patients. Concerning the other main biomarkers analysed in this study, the median plasma concentration of NE and sCD163 observed in our cohort of COVID-19 patients was respectively at least five times higher, and one-point-six times higher, than the values (16-21 ng/ml and 400 ng/ml for NE and sCD163, respectively) reported in recent publications for control groups of 60-70 years old subjects [ 34 , 40 , 41 ]. Our study has some limitations that need to be addressed, most notably its retrospective nature and single-center design.…”

Identifying Neutrophil Extracellular Traps (NETs) in Blood Samples Using Peripheral Smear Autoanalyzers

“…Currently, visual identification of NETs relies on the visualization of DNA, histones, myeloperoxidase, and neutrophil elastase, using advanced immunohistochemical staining and microscopic techniques [ 5 , 9 , 11 , 12 ]. Alternatively, cell-free DNA, MPO-DNA complexes, citrullinated histones C3, neutrophil elastase, and cathelicidin antimicrobial peptides are used as surrogate markers for NETosis [ 13 , 14 , 15 , 16 , 17 , 18 ]. Neutrophil activation marker CD66b has been described as positively correlating with side-fluorescent scatter (SFL) generated by the Sysmex system [ 18 , 19 ], with several reports suggesting that NETs are identified with Sysmex’ SFL scatters [ 18 , 20 ].…”

Clinical significance of neutrophil extracellular traps biomarkers in thrombosis

“…NETs biomarkers have been quantitatively evaluated in VTE patients in several clinical studies [ 44 , 47 – 58 ] (Table 1 ). The levels of plasma DNA [ 53 ], H3Cit-DNA [ 58 ], and NE [ 58 ] were elevated in VTE patients.…”

“…The levels of plasma DNA [ 53 ], H3Cit-DNA [ 58 ], and NE [ 58 ] were elevated in VTE patients. The level of plasma MPO had a good diagnostic performance of VTE [ 59 ], while the diagnostic accuracy of H3Cit-DNA and NE was not superior to that of D-dimer [ 58 ]. On the other hand, the expression of NETs biomarkers may depend on the locations of VTE.…”

“…NETs biomarkers have been quantitatively evaluated in VTE patients in several clinical studies [ 44 , 47 – 58 ] (Table 1 ). The levels of plasma DNA [ 53 ], H3Cit-DNA [ 58 ], and NE [ 58 ] were elevated in VTE patients. The level of plasma MPO had a good diagnostic performance of VTE [ 59 ], while the diagnostic accuracy of H3Cit-DNA and NE was not superior to that of D-dimer [ 58 ].…”