Table 2.
Summary of included studies on human population. Acronyms: POC: prospective observational cohort; RCT: randomised controlled trial; AIS: acute ischemic stroke; aSAH: aneurysmal subarachnoid haemorrhage; TBI: traumatic brain injury; ab: antibody; PAD: protein-arginine deaminase; EdaB: Edaravone Dexborneol; MODS: multiorgan dysfunction syndrome.
| Author | Study | Population | Mechanism of Injury | NET Markers | Methods | Interventions | Outcome | Results |
|---|---|---|---|---|---|---|---|---|
| Gao (2024) [46] |
POC | Adults (n° = 90) | AIS | MPO-DNA complex NE-DNA complex CitH3- DNA complex Nucleosome |
ELISA ELISA CitH3 ELISA kit (Jingmei Biotechnologies, China) human nucleosome ELISA kit (Jingkang Biotech, China) |
Quantification of MPO-DNA, NE-DNA, CitH3-DNA and nucleosome in: -AIS (n° = 45) at hospital admission -healthy controls (n° = 45) |
Difference in NET markers levels | Higher circulating marker levels in AIS (p < 0.0001) |
| Tiwari (2023) [34] |
POC | Adults (n° = 188) |
AIS admitted < 24 h from symptom onset | cfDNA | rtPCR (Quanti Nova probe, Qiagen, Germany) for β-globin gene (Qiagen-Rotor-Gene Q MDX, Qiagen, Germany) |
Quantification of cfDNA in: -AIS (n° = 188) -healthy controls (missing number) |
Association of circulating cfDNA with severity of AIS and poor outcome (mRS ≥ 3 at 3 months) | Levels of cfDNA >10,000 kilogenome eq/L associated with poor outcome (p 0.002); moderate predictive accuracy of cfDNA for stroke prognosis (AUC 0.74) |
| Zhang (2023) [44] |
POC | Adults (n° = 322) |
AIS | CitH3 | ELISA | Quantification of CitH3 at admission in: -severe WMLs (n° = 148) -non severe WMLs (n° = 174) |
Association of circulating CitH3 with severe WMLs (MRI Fazekas score ≥ 3) | Higher citH3 in patients with severe WMLs (45.2 ng/mL [17.8–82.6]) compared to control group (19.6 ng/mL [10.3–46.5]), p = 0.001 |
| Denorme (2022) [18] |
POC | Adults > 18 years old (n° = 54) | AIS admitted ≤ 48 h from symptom onset | CitH3 MPO-DNA complex DNAse I activity |
CitH3 ELISA Kit (Cayman Chemical) In-house-made ELISA kit [C: anti-MPO antibody; D: anti-DNA antibody] DNase I assay kit (Abcam) |
(A) Quantification of CitH3 and MPO-DNA complex in: -AIS (n° = 27) at H admission -healthy controls (n° = 27) (B) DNAse I activity in: -AIS (n° = 27) at H admission -healthy controls (n° = 27) |
Association of NET markers with disability assessed by mRS at discharge | Both CitH3 (r = 0.45, p = 0.024) and MPO-DNA (r = 0.507, p = 0.01) as significant independent variable to predict stroke outcome |
| Grosse (2022) [36] |
POC | Adults (n° = 184) | AIS undergoing thrombectomy | cfDNA DNase I Activity |
Quant-iT™ PicoGreen® assay (Invitrogen, Carlsbad, CA, USA) Fluorometric K429-100 DNase I Activity Assay Kit (BioVision, Milpitas, CA, USA) |
Quantification of cfDNA and DNAse I activity before mechanical thrombectomy in: -AIS with unfavourable outcome (n° = 43) -AIS with favourable outcome (n° = 48) |
Association of NET markers with long-term unfavourable outcome intended as mortality or disability (mRS = 3–6) at 3 months | Baseline cfDNA higher in patients with unfavourable outcome at 90 days (p = 0.044, AUC 0.623); cfDNA at 7 days was higher in those with unfavourable outcome at 90 days (p< 0.001, AUC 0.74); association of cfDNA at admission (p = 0.001, AUC 0.751) and at day 7 (p < 0.001, AUC 0.855) with mortality |
| Huang (2022) [25] |
Non-RCT | adults, 18–85 years old (n° = 45) | AIS clinically diagnosed (NIHSS 3–20) admitted ≤ 48 h from symptom onset | MPO-DNA complex CitH3 |
Cell death detection ELISA kit (Roche, USA) ELISA (Cayman Chemical, USA) |
Quantification of CitH3 and MPO-DNA at admission and at day 3 in: -AIS undergoing EdaB therapy (n° = 15) -AIS undergoing conventional treatment (n° = 15) -healthy donors (n° = 15) |
Effect of Edaravone Dexborneol on NET release and BBB tight junction proteins (occludin) | After stroke onset: higher levels of MPO-DNA and citH3 in AIS (p < 0.01), no significant variability between conventional treatment and Eda.B group. At day 3: significant decrease in MPO-DNA, citH3 in Eda B group (p< 0.01). Strong correlation between NETs and serum occludin levels. |
| Cui (2020) [47] |
POC | Adults (n° = 68) | AIS | cfDNA | BGISEQ-500 sequencer with BWA algorithm | Quantification of cfDNA in: -AIS (n° = 48) -healthy individuals (n° = 20) |
Differences in NET markers levels | Higher circulating cfDNA in AIS (p < 0.05). Prevalence of larger fragments (300–400 bp) rather than smaller fragments (75–250 bp) in AIS. |
| Lim (2020) [48] |
POC | Adults (n° = 81) | AIS | cfDNA DNA-histone complex |
Quant-iT™ PicoGreen® dsDNA kit (Molecular Probes) Cell Death Detection ELISAPLUS (Roche Diagnostics GmbH) |
Quantification of cfDNA and DNA-histone in: -AIS (n° = 58) -healthy controls (n° = 23) |
NET makers in AIS population and association of circulating cDNA levels with MACE incidence Define sensitivity of cDNA for AIS |
Significant increase in circulating cfDNA in AIS, but not for DNA-histone. Higher incidence of MACE in AIS group but not statistically significant. High sensitivity of cfDNA for AIS (AUC 0.859). |
| Vajpeyee (2020) [38] |
POC | Adults (n° = 54) | stroke-like symptoms with samples withdrawn ≤ 12 h from onset | cfDNA | rtPCR (Quanti Nova probe, Qiagen, Germany) n) for β-globin gene (Qiagen-Rotor-Gene Q MDX, Qiagen, Germany) | Quantification of cfDNA at admission in: -AIS with unfavourable outcome (missing number) -AIS with favourable outcome (missing number) |
CfDNA as early marker of AIS severity and poor prognosis (mRS ≥3 at 3 months) | Higher circulating cfDNA in patients with poor outcome (p < 0.05). Favourable outcome in patients undergoing thrombolysis or mechanical thrombectomy (n° = 26), who presented cfDNA level <10,000 kilogenome-eq/L at admission (p < 0.05). |
| Valles (2017) [49] |
POC | Adults (n° = 270) | AIS admitted ≤ 24 h from symptom onset | cfDNA Nucleoome CitH3 |
fluoroscopy with Sytox Green (Invitrogen, Carlsbad, CA, USA) Cell death detection ELISAPLUS (Roche Diagnostics, Madrid, Spain) Cell death detection ELISAPLUS (Roche Diagnostics, Madrid, Spain) |
Quantification of cfDNA, nucleosome and CitH3 at admission in: -AIS (n° = 243) -healthy controls (n° = 27) |
Differences in NETosis markers levels and their association with clinical characteristics, stroke severity and outcome at 1 year | Elevated circulating NET markers in AIS at admission; higher in those with NIHSS >14. At discharge, all the markers positively correlated with NIHSS score (≥6) and mRS score (>2); at multivariate analysis, citH3 was associated with AF and all-causes mortality at 1 year. |
| Cao (2023) [40] |
POC | Adults (n° = 20) | Severe TBI at admission | cfDNA CitH3-DNA complex |
Quant-iT PicoGreen dsDNA Assay kit (Invitrogen, USA) Cell Death ELISA (Roche, Basel, Switzerland) |
Quantification of cfDNA and CitH3 at admission in: -TBI (n° = 10) -Healthy matched controls (n° = 10) |
Association of NET markers with worse neurological function intended as low GCS and elevated ICP | Levels of cfDNA were significantly elevated in TBI; cfDNA inversely correlated to GCS and positively correlated to ICP. |
| Mi (2023) [50] |
POC | Adults (n° = 16) | TBI | cfDNA | Quant-iT™ PicoGreen® dsDNA assay kit (Invitrogen, Carlsbad, CA, USA) | Quantification of cfDNA at admission in: -TBI (n° = 8) -healthy control (n° = 8) |
Differences in NETosis markers levels | Higher circulating cfDNA in TBI (p < 0.05) |
| Ben Zvi (2022) [51] |
POC | Adults, 18–67 years old (n° = 60) | Isolated mild TBI with no pathological findings at head CT scan | cfDNA | Fast SYBR Gold assay + fluorescence 96-well fluorimeter (SpectraMax Paradigm, Molecular Devices) | (A) Quantification of cfDNA in: -m TBI at admission (n° = 30) -matched control (n° = 30) (B) Correlation of cfDNA at admission in: -Survivors with moderate/severe impairment (n° = 4) -Survivors with mild or no impairment (n° = 14) |
cfDNA as a prognostic marker for post-concussion syndrome assessed by CCT 1 and CCT 2 at 3 months | Higher circulating cfDNA at admission in patients with moderate/severe cognitive impairment according to CTT 1 |
| Hazeldine (2021) [42] |
POC | Adults (n° = 155) | TBI | cfDNA DNase I Activity |
fluoroscopy ELISA (LifeSpan BioSciences Inc., UK) |
Quantification of cfDNA and DNAse I activity in: -Isolated TBI (n° = 21) TBI + extracranial injuries (n° = 53) -Extracranial injury (n° = 81) -Healthy control (n° = 75) |
Association of cfDNA with MODS defined as SOFA score ≥ 6 for more than 2 days | Significantly higher circulating cfDNA in all trauma patients at all post-injury time points; at 48–72 h, higher cfDNA in TBI patients developing MODS (p < 0.05); in all patients, significant reduction of DNAse activity and increase in DNAse I at 1 h, 4–12 h, 48–72 h (p < 0.005) |
| Marcatti (2021) [32] |
POC | Adults (n° = 93) | TBI requiring activation of Trauma Team | cfDNA | Quant-iT™ PicoGreen® dsDNA assay kit (Invitrogen, Carlsbad, CA, USA) | Quantification of cfDNA at admission in: -sTBI (n° = 33) -mTBI (n° = 20) -healthy volunteers (n° = 20) |
Association of cfDNA with TBI severity | Higher circulating cfDNA in TBI (p < 0.0001); most of cfDNA had mitochondrial origin. |
| Shaked (2014) [33] |
POC | Adults (n° = 58) | Blunt isolated TBI admitted ≤ 4 h from event | cfDNA | fluoroscopy with fluorochrome SYBR Gold | (Quantification of cfDNA at admission in: -TBI (n° = 28) -Healthy controls (n° = 30) |
Association of cfDNA with severity (GCS at admission), disability (GOS at hospital discharge) and mortality | cfDNA levels were significantly higher in non survivors, in severe TBI and in patients with disability |
| Moraes Rodrigues Filho (2014) [39] |
POC | Adults > 16 years (n° = 213) |
Severe TBI | cfDNA | rtPCR (Life Technologies, Carlsbad, CA, USA) for β-globin gene | (A) Quantification of cfDNA at 12 h from ICU admission in: -severe TBI (n° = 188) -healthy control (n° = 25) (B) Comparison of cfDNA levels at 12 h from ICU admission in: -non-survivors (n° = 66) -survivors (n° = 122) |
Association of cfDNA and mortality after ICU admission | Higher circulating cfDNA as an independent variable associated with mortality (p <0.001): cut-off level of 171,381 kilogen eq/L measured 12 h after entry predicting fatal outcome with high accuracy (AUC 0.9). Correlation of higher DNA levels and lower GCS at admission (p = 0.001) |
| Macher (2012) [35] |
POC | Adults (n° = 65) | Severe TBI | cfDNA | rtPCR | (A) Quantification of cfDNA in: -severe TBI (n° = 65) at admission, at day 1 and day 3 -healthy controls (missing number) (B) Quantification of cfDNA decrease ratio at 24 h, 48 h and 72 h in: -non-survivors (n° = 14) -survivors (n° = 51) |
Differences in cfDNA markers levels and association with mortality | Higher cfDNA in TBI at admission, reduction at 24 h and 48 h and mild increase at 72 h; significant decrease of cfDNA within 24 h from injury in survivors. Correlation of cfDNA levels with GCS at admission and at 24 h (p < 0.05) |
| Campello Yurgel (2007) [37] |
POC | Male adults (n° = 54) | Severe TBI admitted to ICU ≤ 24 h from injury | cfDNA | rtPCR for β-globin with Taqman system | (A) Quantification of cfDNA at admission and at 24 h in: -severe TBI (n° = 41) -healthy male volunteers (n° = 13) |
cfDNA as a prognostic marker for mortality | Higher cfDNA at 24 h from admission significantly correlated to mortality (r = 0.356, p < 0.03); cut-off of 77,883.5 kilog-eq/L at 24 h predictive for fatal outcome (ROC curveAUC 0.71). |
| Witsch (2022) [43] |
POC | Adults (n° = 78) | aSAH confirmed at CT scan | MPO-DNA complex | Cell Death Detection ELISA (Roche, Basel, Switzerland) |
(A) Quantification of MPO-DNA complex in aSAH population (n° = 78) at admission and at day 4 (B) Comparison of MPO-DNA complex levels in: -aSAH with DCI (n° = 17) -aSAH without DCI (n° = 12) (C) Comparison of MPO-DNA complex levels in: -aSAH with VAS (n° = 17) -aSAH without VAS (n° = 12) |
Association of MPO-DNA complex levels with DCI | Significant reduction in MPO-DNA levels at day 4 compared to baseline. Significant increase in MPO-DNA levels in DCI (p = 0.04) compared to those without DCI. Significant reduction in MPO-DNA levels in patients with vasospasm (p =0.006) compared to patients without vasospasm. |
| Zeng (2021) [41] |
POC | Adults (n° = 20) | aneurysmal SAH ≤ 24 h from admission | CitH3 | Cell death detection ELISA PLUS (Roche) [C: mouse anti-histone biotinylated antibodies-D: anti CitH3 antibodies] |
Quantification of CitH3 at admission in: -aSAH (n° = 10) -healthy controls (n° = 10) |
Association of CitH3 levels with aSAH severity assessed by Hunt and Hess classification | Significant increase of CitH3 in aSAH (p = 0.0012); linear correlation between plasma CitH3 and Hunt and Hess classification (p = 0.0118) |