Circulating NETosis biomarkers predict severity, 12-month functional outcome, and survival after spontaneous intracerebral hemorrhage

Background Spontaneous intracerebral hemorrhage (ICH) carries high early mortality and long-term disability; however, circulating biomarkers that capture both bleeding severity and prognosis are lacking. Neutrophil extracellular trap formation (NETosis), quantifiable in plasma as citrullinated histone H3 (H3Cit), cell-free DNA (cfDNA), and nucleosomes, is implicated in secondary brain injury after ICH. Methods Three hundred fifteen adults with first-ever spontaneous ICH within 24 h of onset and 160 community controls were enrolled. Plasma H3Cit, cfDNA and nucleosomes, admission hematoma volume, NIHSS, and GCS were recorded; the modified Rankin Scale (mRS) was assessed at 12 months. NETosis biomarkers entered linear, logistic, and Cox regressions as log2-transformed continuous variables and were adjusted progressively for time-to-sampling, demographics, vascular risk factors, surgical treatment, NIHSS, and hematoma volume. Incremental predictive value over a clinical baseline model was assessed using area under the receiver operating characteristic curve (AUC)/concordance index (C-index), continuous net reclassification improvement (NRI), and integrated discrimination improvement (IDI). Results All three biomarkers were higher in ICH than controls (all p < 0.001) and were several-fold higher in 12-month decedents than survivors, despite no difference in absolute neutrophil count. After adjustment, H3Cit was independently associated with hematoma volume, NIHSS and GCS; cfDNA and nucleosomes were independently associated with hematoma volume. After full adjustment for demographics, vascular risk factors, surgical treatment, NIHSS, and hematoma volume, each doubling of H3Cit (OR: 1.577, 95% CI: 1.368–1.838), cfDNA (OR: 1.687, 95% CI: 1.259–2.291), and nucleosomes (OR: 1.622, 95% CI: 1.297–2.057) independently predicted 12-month mRS ≥ 3 (all p < 0.001); per doubling, H3Cit (HR: 1.358, 95% CI: 1.207–1.528), cfDNA (HR: 1.619, 95% CI: 1.287–2.038), and nucleosomes (HR: 1.568, 95% CI: 1.310–1.877) independently predicted 12-month death. Adding H3Cit raised the AUC for mRS ≥ 3 from 0.814 to 0.869 (ΔAUC: 0.055, p < 0.001) and the C-index for death from 0.765 to 0.818 (ΔC: 0.052, p < 0.001); for cfDNA and nucleosomes, the gains in discrimination were not significant, although NRI and IDI improved. Conclusion Plasma H3Cit, cfDNA, and nucleosomes within 24 h of ICH track hemorrhage severity and predict 12-month mRS ≥ 3 and death beyond established clinical scores, with H3Cit showing the most consistent incremental value.