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.

Keywords: clot lysis; hemorrhagic stroke; intracerebral hemorrhage; neutrophil extracellular traps; outcome.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2021 Orbán-Kálmándi, Árokszállási, Fekete, Fekete, Héja, Tóth, Sarkady, Csiba and Bagoly.

Figures

Figure 1

Associations of the 10%CLT parameter of the modified clot lysis assay (mCLA) with stroke severity on admission (A), ICH score calculated on admission (B), survival by day 14 (C), and the modified Rankin Scale (mRS) on day 90 (D). mCLA is performed in the presence of cell-free DNA and histones. Box and whisker plots indicate median, interquartile range, and total range. 10% CLT: 10% clot lysis time, ICH, intracerebral hemorrhage; NIHSS, National Institutes of Health Stroke Scale; mRS, modified Rankin Scale. *p < 0.05.

Figure 2

Associations of the estimated cerebral hematoma volume as calculated from on admission CT scans with patient survival by day 14 (A), and the modified Rankin Scale (mRS) on day 90 (B). Box and whisker plots indicate median, interquartile range, and total range. mRS, modified Rankin Scale. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Correlation between the modified clot lysis assay (mCLA) parameters and estimated intracerebral hematoma volume of the ICH patients. mCLA is performed in the presence of cell-free DNA and histones. Correlation between estimated intracerebral hemorrhage volume and time to maximal absorbance parameter (A), 10% clot lysis time (10%CLT) (B), and clot lysis assay area under the curve (CLA AUC) (C) are depicted.

Figure 4

Receiver operator characteristic (ROC) curves of the modified clot lysis assay (mCLA) parameters for predicting long-term functional outcomes (mRS 0–1 vs. 2–6) of intracerebral hemorrhage stroke patients. mCLA is performed in the presence of cell-free DNA and histones. ROC curve and descriptive statistics including best cut-off value as determined by the Youden index are depicted for time to maximal absorbance parameter (A), 10% clot lysis time (10%CLT) (B), 50% clot lysis time (50%CLT) (C).

Figure 5

Kaplan-Meier survival curves of patients with spontaneous intracerebral hemorrhage according to the result of the modified clot lysis assay on admission (10%CLT below and above the limit of 38.5 min).