A Modified in vitro Clot Lysis Assay Predicts Outcomes in Non-traumatic Intracerebral Hemorrhage Stroke Patients—The IRONHEART Study

Abstract

Background: Non-traumatic intracerebral hemorrhage (ICH) accounts for 10–15% of all strokes and results in a higher rate of mortality as compared to ischemic strokes. In the IRONHEART study, we aimed to find out whether a modified in vitro clot lysis assay method, that includes the effect of neutrophil extracellular traps (NETs) might predict ICH outcomes.Patients and Methods: In this prospective, observational study, 89 consecutive non-traumatic ICH patients were enrolled. Exclusion criteria included aneurysm rupture, cancer, liver- or kidney failure or hemorrhagic diathesis. On admission, detailed clinical and laboratory investigations were performed. ICH volume was estimated based on CT performed on admission, day 14 and 90. A conventional in vitro clot lysis assay (CLA) and a modified CLA (mCLA) including cell-free-DNA and histones were performed from stored platelet-free plasma taken on admission. Clot formation and lysis in case of both assays were defined using the following variables calculated from the turbidimetric curves: maximum absorbance, time to maximum absorbance, clot lysis times (CLT) and area under the curve (CLA AUC). Long-term ICH outcomes were defined 90 days post-event by the modified Rankin Scale (mRS). All patients or relatives provided written informed consent.Results: Patients with more severe stroke (NIHSS>10) presented significantly shorter clot lysis times of the mCLA in the presence of DNA and histone as compared to patients with milder stroke [10%CLT: NIHSS 0–10: median 31.5 (IQR: 21.0–40.0) min vs. NIHSS>10: 24 (18–31.0) min, p = 0.032]. Shorter clot lysis times of the mCLA showed significant association with non-survival by day 14 and with unfavorable long-term outcomes [mRS 0–1: 36.0 (22.5.0–51.0) min; mRS 2–5: 23.5 (18.0–36.0) min and mRS 6: 22.5 (18.0–30.5) min, p = 0.027]. Estimated ICH volume showed significant negative correlation with mCLA parameters, including 10%CLT (r = −0.3050, p = 0.009). ROC analysis proved good diagnostic performance of mCLA for predicting poor long-term outcomes [AUC: 0.73 (0.57–0.89)]. In a Kaplan-Meier survival analysis, those patients who presented with an mCLA 10%CLT result of >38.5 min on admission showed significantly better survival as compared to those with shorter clot lysis results (p=0.010).Conclusion: Parameters of mCLA correlate with ICH bleeding volume and might be useful to predict ICH outcomes.

Neurophysiological Examinations as Adjunctive Tool to Imaging Techniques in Spontaneous Intracerebral Hemorrhage: IRONHEART Study

“…Currently, only on-admission blood glucose levels and INR values were analyzed. Detailed hemostasis, renal function, white blood cells, hemoglobin, hematocrit, and platelet count data were described in our previous article ( 8 ).…”

“…A conventional in vitro clot lysis assay (CLA) and a modified CLA (mCLA)—including cell-free DNA and histones—were performed from stored platelet-free plasma, taken on admission. Parameters of mCLA correlated with ICH bleeding volume ( 8 ).…”

Role of the Extracellular Traps in Central Nervous System

“…A recent research reported the NET infiltration in the brain of patients who died from spontaneous intracerebral hemorrhage (sICH), suggesting that NETs might interact with early hemostasis within the hematoma core and with the surrounding neuroinflammatory response (57). Orbań-Kaĺmańdi et al pointed out that a modified clot lysis assay, incorporating the effect of NETs, could suggest unfavorable outcomes in spontaneous, nontraumatic ICH based on a prospective observational study data (58). Also, in subarachnoid hemorrhage (SAH), the current studies revealed that NETs can induce the alteration of microglia into a proinflammation subtype in order to promote neuroinflammation and cause adverse consequences, suggesting that NETs may be a potential target for the treatment SAH in its early phase (59,60).…”

“…Orbán-Kálmándi et al. pointed out that a modified clot lysis assay, incorporating the effect of NETs, could suggest unfavorable outcomes in spontaneous, nontraumatic ICH based on a prospective observational study data ( 58 ). Also, in subarachnoid hemorrhage (SAH), the current studies revealed that NETs can induce the alteration of microglia into a proinflammation subtype in order to promote neuroinflammation and cause adverse consequences, suggesting that NETs may be a potential target for the treatment SAH in its early phase ( 59 , 60 ).…”