Neutrophil‐to‐lymphocyte ratio and its changes predict the 3‐month outcome and mortality in acute ischemic stroke patients after intravenous thrombolysis

Abstract Background and purpose The neutrophil‐to‐lymphocyte ratio (NLR) has been demonstrated as a prognostic inflammatory biomarker in ischemic stroke. The study aimed to investigate the association of NLR and its dynamic change with long‐term outcome and mortality in acute ischemic stroke (AIS) patients who received intravenous thrombolysis (IVT). Methods From a prospective cohort, AIS patients receiving IVT (alteplase, 0.9 mg/kg) with complete NLR data were retrospectively screened. Based on 3‐month modified Rankin scale score (mRS), patients were classified into good group (mRS 0–1) and poor outcome group (mRS 2–6), or survival group (mRS 0–5) and death group (mRS 6). Multivariate logistic regression analysis and receiver operating curve were used to identify prognostic factors and their predictive powers. Results A total of 259 eligible patients were enrolled in our study. Logistic regression analysis showed that NLR at 24 h (adjusted odds ratio [aOR] 1.182), 12 days (aOR 1.218) after IVT was independent predictors of 3‐month outcome with the AUC of 0.815, 0.820, respectively, whereas NLR at 24 h (aOR 1.17), 12 days (aOR 1.252) after IVT and percentage changes of NLR between admission and 24 h after IVT (aOR 1.214), and between admission and 12 days after IVT (aOR 1.233) were independent predictors of 3‐month mortality with the AUCs of 0.86, 0.902, 0.814, and 0.855, respectively. Conclusion The comprehensive report suggests that NLR and its dynamic changes are associated with 3‐month outcome and mortality in AIS patients after IVT with good predictive powers.


INTRODUCTION
Based on data from the Global Burden of Disease Study 2019 (GBD 2019Stroke Collaborators, 2021, stroke is the second leading cause of disability and mortality worldwide, and the prevalence of stroke in China has continued to increase during 2013−2019 (Tu et al., 2022).
As the most common type of stroke, acute ischemic stroke (AIS) possesses high morbidity and mortality (GBD 2019Stroke Collaborators, 2021Tu et al., 2022). Intravenous thrombolysis (IVT) is one of effective treatments for AIS, but about 1/3 patients get good prognosis (Kastrup et al., 2017). It has been a hot topic about how to accurately predict the outcome of AIS patients after IVT. A lot of prognostic factors have been identified, for example, advanced age (Emberson et al., 2014), higher National Institutes of Health Stroke Scale (NIHSS) score (Kawiorski et al., 2016), and neuroinflammatory response (Kim et al., 2016;Parikh et al., 2020;Ramiro et al., 2018) are associated with poor outcome after thrombolysis in stroke patients. Due to its low cost and easy availability in clinical practice, neutrophil-to-lymphocyte ratio (NLR) has been reported to have strong prognostic effects on clinical outcomes of AIS patients without reperfusion treatment, for example, the association of NLR with short-term mortality, stroke severity on admission, unfavorable functional outcome, and recurrent ischemic stroke (Goyal et al., 2018;Sharma et al., 2022;Tokgoz et al., 2013;Wang et al., 2019). In AIS patients treated with IVT, most of these studies focused on the predictive effects of NLR at admission and within 24 h after IVT (Brooks et al., 2014;Li et al., 2022;Maestrini et al., 2015), and only a few studies expanded the time points to 48 h, 3 days, or even 7 days (Guo et al., 2016;Weng et al., 2021); however, there is a lack of studies to systemically investigate the prognostic roles of NLR at extended time point and their dynamic changes in AIS patients after IVT.
In this study, we aimed to investigate the association of NLR at admission, 24 h and 12 days after IVT and their dynamic changes with 3-month outcome and mortality in AIS patients after IVT.

Outcome assessments
All patients were assessed at 3-month using mRS. Good outcome was defined as a functional independence (a mRS score of 0 or 1), whereas a score of 2 or greater on the mRS was defined as poor outcome. Survival was defined as a score of 1-5 on the mRS, whereas a score of 6 on the mRS was defined as death group.   Table 1. There were 175 males (67.6%); the median admission NIHSS was 7 (IQR 4-14) and 108 (69.2%) with good outcome. Compared with good outcome group, patients with poor outcome had higher NIHSS score and lower weight and body mass index, more prevalence of atrial fibrillation (all p < .05, Table 1). Neutrophil and lymphocyte at 24 h and 12 days after IVT, NLR2, NLR3, cNLR1-2, and cNLR1-3 were higher in poor versus good outcome group (all p < .001, Table 2). Compared with survival group, patients with death group had higher NIHSS score and heart rate, more prevalence of atrial fibrillation (all p < .05, Table 1). Neutrophil and lymphocyte at 24 h and 12 days after IVT, NLR2, NLR3, cNLR1-2, and cNLR1-3 were higher in survival versus death group (all p < .05, Table 2). Furthermore, cNLR1-2 was most obvious change among three time points, whereas cNLR1-2 and cNLR1-3 were significantly associated with poor outcome or death ( Figure 5).

DISCUSSION
In this study, we found that (1) there were dynamic changes of NLR in AIS after IVT; (2) NLR at 24 h and 12 days after IVT, but not at the baseline, as well as its dynamic changes were independent prognostic factors for poor outcome and death; (3) NLR and its dynamic changes have good predicting power for poor outcome and death in this population. Collectively, this is the first comprehensive study to investigate the association of NLR and its dynamic changes with outcome and death in AIS after IVT and provide evidence of their good predictive power for outcome and death.
It is already known that inflammation is implicated in the pathogenesis of stroke (Kim et al., 2016;Parikh et al., 2020;Ramiro et al., 2018). Neutrophils have been demonstrated to aggravate brain damage by releasing inflammatory mediators, aggravating oxidative stress, and increasing blood-brain barrier permeability ( (c) Showing the dynamic changes of NLR levels between survival and death groups. NLR, neutrophil-to-lymphocyte ratio; time1, admission; time2, 24 h after intravenous thrombolysis; time3, 12 ± 2 days after intravenous thrombolysis.

TA B L E 2
Comparison of inflammatory biomarkers between good and poor outcomes (or survival and death).

F I G U R E 5
Comparison of the changes in neutrophil-to-lymphocyte ratio (NLR) at different time points between groups. (a) Comparison among the cNLR1-2, cNLR1-3 and cNLR2-3. (b) Comparison of the changes in NLR at different time points between good and poor outcome groups. (c) Comparison of the changes in NLR at different time points between survival and death groups. cNLR1-2, percentage change of NLR between admission and 24 h after intravenous thrombolysis; cNLR1-3, percentage change of NLR between admission and 12 ± 2 days after intravenous thrombolysis; cNLR2-3, percentage change between 24 h and 12 ± 2 days after intravenous thrombolysis.

TA B L E 3
Univariate analysis of predictors associated with 3-month poor outcome or death. Abbreviations: CE, cardioembolism; CI: confidence interval; cNLR1-2, percentage change between NLR1 and NLR2; cNLR1-3, percentage change between NLR1 and NLR3; cNLR2-3, percentage change between NLR2 and NLR3; DBP, diastolic blood pressure; IVT, intravenous thrombolysis; LAA, large artery atherosclerosis; NIHSS, National Institute of Health Stroke Scale; OR, odds ratio; SAO, small-artery occlusion; SBP, systolic blood pressure; SOE, stroke of other determined etiology; SUE, stroke of undetermined etiology; TOAST, Trial of Org 10 172 in acute stroke treatment. a p for trend. the brain injury and peak at 24-48 h, followed by a gradual reduction (Alam et al., 2020). Consistent with previous findings, our study showed that neutrophil levels were at their peak at 24 h after IVT and fell back at 12 days after IVT, and its increase was closely related to their 3month poor outcome and death. The lymphocyte counts as an index for general health, influenced by acute physiologic stress, were actively involved in a protective mechanism in the ischemic brain (Li et al., 2015;Liesz et al., 2013). The decrease in the lymphocyte count is related to the early neuroinflammation and the deterioration of immune func-tion, leading to poor clinical outcomes (Yang et al., 2020). Consistent with these studies, we found that the lymphocytes had a significant decrease at 24 h after IVT, which gradually recovered at 12 days after IVT, and its decrease was associated with poor outcome and death.

Poor outcome Death
NLRs, calculated from neutrophils and lymphocytes, were found to be closely related to the effects of neutrophils and lymphocytes on stroke and their interaction mechanisms (Brooks et al., 2014;Li et al., 2022;Parikh et al., 2020;Ramiro et al., 2018). Prior studies showed that NLR was used to predict the prognosis of patients with AIS after TA B L E 4 Multivariate analysis of predictors associated with 3-month poor outcome or death. thrombolysis in stroke patients (Brooks et al., 2014;Li et al., 2022;Maestrini et al., 2015). Most studies have collected the NLR value at several time points within 7 days after IVT (generally at admission, 24, 48 h, and 7 days) (Chu et al., 2020;Guo et al., 2016) and found that NLR level increased after stroke onset and reached the peak at 12-48 h (Li et al., 2022;Sadeghi et al., 2022;Shi et al., 2018;Weng et al., 2021). In agreement with these studies, we found that NLR peaked at 24 h after IVT. Furthermore, we found that NLR returned to baseline levels around 12 days after IVT, which was reported previously. In addi-tion, we found NLR 24 h and 12 days after IVT and its change from the baseline are closely related to the 3-month outcome and death of AIS patients.

Poor outcome Death
The strength of this study was to comprehensively explore the association of NLR dynamic change with clinical outcome in patients with AIS after IVT based on a prospective cohort. We found that NLR changes between admission and 24 h after IVT, and admission and 12 days after IVT are closely related to the 3-month outcome and mortality of AIS patients. Furthermore, we provided the optimal cutoff of NLR F I G U R E 7 Receiver operating characteristic curve (ROC) analysis of inflammatory biomarkers to predict 3-month death (A, unadjusted; B, adjusted). Adjusted by age, body mass index, heart rate, admission National institutes of Health Stroke Scale (NIHSS) score, TOAST subtype. AUC, area under the curve; CI, confidence interval; NLR, neutrophil-to-lymphocyte ratio; NLR2, NLR of 24 h after intravenous thrombolysis; NLR3, NLR of 12 ± 2 days after intravenous thrombolysis; cNLR1-2, percentage change between NLR1 and NLR2; cNLR1-3, percentage change between NLR1 and NLR3.
at 24 h, 12 days after IVT to predict 3-month poor outcome and mortality, which may assist in outcome stratification in this population and future clinical trial design.

Limitations
Our study has several limitations. First, the main limitation is the retrospective nature of analysis, which inevitably resulted in the selection bias and confounding factors. Second, only 259 patients were included from about 40 centers in the current study. The relatively small sample may introduce a potential selection bias. Third, only patients who received IVT and collected blood routine examination at three time points were included in our analysis, so we were unable to investigate the effect of NLR in different populations with IVT, endovascular or standard stroke care without reperfusion treatment, and compare the predictive value of NLR versus other inflammatory biomarkers, such as CRP, IL-6, and TNF-alpha, in this population. Thus, the findings warrant further confirmation in prospective trials with large sample.

CONCLUSION
This comprehensive study showed that NLR at 24 h and 12 days after IVT, and its dynamic change were independent prognostic factors for 90-day clinical outcomes in AIS patients after IVT with good predictive powers.

AUTHOR CONTRIBUTIONS
Hui-Sheng Chen supervised the design. Qiong Wu conducted the analyses and drafted the manuscript. Hui-Sheng Chen critically revised the manuscript.