Red cell index: A novel biomarker for 3‐month mortality in acute ischemic stroke patients treated with intravenous thrombolysis

Abstract Background The red cell index (RCI) was described as a biomarker for evaluating respiratory function in previous studies, but the relationship between RCI and stroke, remained a mystery. The present study aimed to probe the association between RCI at 24‐hr and 3‐month mortality and functional outcomes among acute ischemic stroke (AIS) patients treated with recombinant tissue plasminogen activator (r‐tPA). Methods A total of 217 AIS patients between January 2016 and January 2019 were recruited in this retrospective study. AIS patients were grouped in terms of RCI tertiles. Predictive factors were confirmed via multivariate logistic regression analysis. The receiver operating characteristic (ROC) was used to assess the ability of RCI in predicting mortality. In addition, the risk of 3‐month all‐cause mortality was evaluated by Cox proportional hazard model. Results We grouped AIS patients into tertiles with the purpose of comparing clinical factors and RCI levels. Multivariate logistic regression analysis presented that RCI (odds ratio [OR] = 1.443, 95% confidence interval [CI] [1.167–1.786], p = 0.001) was an independent biomarker for 3‐month all‐cause mortality. The best cutoff value of RCI was 2.41 (area under the curve [AUC] = 0.639, 95% CI [0.501–0.778], p = .032), with a sensitivity of 40.9% and a specificity of 89.7%. Cox survival analysis demonstrated a positive significant correlation between RCI (hazard ratio [HR] = 1.332, 95% CI [1.148–1.545], p < .001) and mortality risk. Conclusion RCI, a potential predictor, was significantly associated with 3‐month mortality in AIS patients with r‐tPA.

globin levels were associated with the risk of hypertension, blood pressure, and carotid atherosclerosis, while low ones were related to the risk of cardiovascular death and major bleeds, which could probably enlarge infarct size and accelerate infarct growth (Atsma et al., 2012;Kalra et al., 2017). There were relationships between hemoglobin treated with mechanical thrombectomy or arterial thrombolysis and prognosis (Ling et al., 2020;Sun et al., 2020). Moreover, a recent article had demonstrated that elevated hemoglobin levels in acute phase were associated with poor outcomes at 3 months after ischemic stroke, which further illustrated the association between the three: hemoglobin, prognosis, and mortality (Guo et al., 2019).
Inflammation and the immune response that were also key elements in stroke played a significant role in the pathogenesis of ischemic stroke and its subtypes, leading to infarct progression through reperfusion injury though under the treatment of thrombolysis or thrombectomy. The immune system was involved in the brain damage caused by ischemia, while the damaged brain played an immunosuppressive role, contributing to deadly infections. The inflammation system was involved in all phases of the ischemic cascade, from early injury events to late regeneration (Iadecola & Anrather, 2011).
Lymphocyte counts provided insight into the inflammatory process and reflected the effect of acute physiological stress. Lower lymphocyte counts were significantly associated with the poor functional outcome with increased probability (Kim et al., 2012). Relative decreased lymphocyte may mirror cortico-related stress responses and sympathetic tone, resulting in the increasing production of proinflammatory cytokines and eventually increased the severity of ischemic injury (Acanfora et al., 2001). The activation and aggregation of platelet, a well-known hemostatic functional hemocyte variable, not only played an intermediary role in thrombus formation, but also participated in the inflammatory process (Hvas, 2016), giving rise to the instability of plaque. Accumulated evidence indicated that a low platelet level was associated with the poor outcome of AIS patients (Mayda-Domac et al., 2010). As for erythrocytes, during ischemic stroke, red blood cells went through oxidative and hydrolytic changes, also causing inflammatory processes and changes of cellular rheology. The latest articles manifested that alterations in the RBC membrane and dense matted deposits (DMDs) that caused blood cells to be trapped in the mesh played an essential part in the presence of thrombi, which had ulteriorly impacts on ischemic stroke (Pretorius & Lipinski, 2013).
Currently, a new indicator, the red cell index (RCI), has been put forward, taking hemoglobin (Hb), lymphocyte (Lym), platelet (Plt), F I G U R E 1 Flow diagram showing the patient selection process and red blood cell (RBC) into regard, and was calculated using the following equation: (RBC ×Hb) / (Lym ×Plt) (Guang et al., 2018).
Theoretically, the RCI level was inversely proportional to the respiratory function. Meanwhile, RCI could be deemed as a simple and resultful biomarker for evaluating respiratory function (Guang et al., 2018), but no studies mentioned the relationship between RCI and prognosis of ischemic stroke. To our knowledge, the prognostic value of RCI in AIS patients with r-tPA infusion was discussed herein for the first time.
The present study aimed to evaluate the correlation between RCI at 24-hr and 3-month mortality, and functional outcomes among AIS patients treated with r-tPA.

| Study population
As demonstrated in Figure 1 Exclusion criteria were as follows: (1) a bridging therapy consisting of intravenous r-tPA followed by endovascular therapy; (2) a therapy of urokinase thrombolysis; (3) severe liver or kidney dysfunction; (4) autoimmune diseases; (5) chronic inflammatory diseases or malignant tumor; (6) acute myocardial infarction; and (7) incomplete data. Finally, 217 patients were included in the current study.
The study was approved by the Ethics Committee of the Third Affiliated Hospital of Wenzhou Medical University and was carried out in accordance with the Declaration of Helsinki. The Ethics Committee number for the study is YJ2020034. All patients or their relatives gave informed consent in writing.

| Data collection
Demographic characteristics (age and sex), baseline vital signs (systolic blood pressure and diastolic blood pressure), baseline stroke risk factors (hypertension, diabetes mellitus, hyperlipemia, history of stroke and smoking, atrial fibrillation, and coronary heart disease) and the National Institutes of Health Stroke Scale (NIHSS) on admission and at 24 hr were reviewed from the database. In addition, we recorded 3-month modified Rankin Scale (mRS) after onset of AIS via two trained physicians on phone interview.

| Study endpoints
3-month all-cause mortality was the primary endpoint. The secondary endpoint was the functional outcomes evaluated by the mRS: a favorable outcome was defined as mRS ≤2, while an unfavorable outcome was defined as mRS >2. The severity of AIS was defined according to NIHSS scores, and thus, AIS-population was grouped as follows: mild or moderate stroke (NIHSS scores: 0-10) and severe stroke (NIHSS scores: > 10) (Asberg et al., 2018). According to the cutoff point, the RCI level was dichotomized at high or low values while involved in the survival analysis. Cox multivariate proportional hazards regression analysis was used for assessing the risk of a future clinical event in an individual patient. A p <.05 was considered significant for all analyses.

| Characteristics of study subjects
The baseline demographic and clinical characteristics of eligible patients were summarized in Table 1

| Association between RCI values and clinical prognosis
The distribution of mRS scores at 3 months and NIHSS at 24 hr in patients treated with thrombolysis were presented in Figure 2.
Patients with higher RCI level tended to have higher stroke severity and poor prognosis at 3 months significantly. To research the factors that could predict the outcome of poor function, univariate and multivariate logistic regression analysis were conducted (
ROC curve was performed to evaluate the ability of RCI, platelet to lymphocyte ratio (PLR), and monocyte to lymphocyte ratio (MLR) Xu et al., 2019) in predicting the 3-month mortality in AIS patients (Figure 3) Abbreviations: AF, atrial fibrillation; CHD, coronary heart disease; DBP, diastolic blood pressure; NIHSS, National Institutes of Health Stroke Scale; RCI, red cell indexSBP, systolic blood pressure.

TA B L E 2
The logistic regression analyses of predictors to unfavorable functional outcome in 3 months regression proportional hazard model analyses were further performed after adjusting the feasible confounding effect, showing a significant association between RCI (hazard ratio [HR] = 1.332, 95% CI [1.148-1.545], p <.001) and mortality risk (Table 4).

| D ISCUSS I ON
RCI was calculated using the equation: (RBC ×Hb) / (Lym ×Plt), taking four key indicators: RBC, Hb, Lym, and PLT. In this retrospective study, AIS patients with lower RCI values, treated with r-tPA thrombolysis, had higher lymphocyte and platelet counts. And our data presented that RCI was significantly associated with 3-month mortality. ROC curve further confirmed RCI level was an effective biomarker to predict 3-month mortality. In addition, there was a positive correlation between RCI level and mortality risk, even after adjusting the potential confounding effect.
Hemoglobin, a well-established marker to detect anemia, had been suggested to have possible pathophysiological pathways. The damage of endothelial cell could upgrade growth factor level, which strengthened hematopoiesis level (Wisniewski et al., 2020). And elevated blood pressure was prevalent in the acute period of ischemic stroke (Qureshi et al., 2007), activating the renin-angiotensin-aldosterone system and in turn causing the production of angiotensin-2, vasoconstriction, and erythropoietin. In addition to the elevation of blood pressure, endothelial cell can also raise the hemoglobin level. Besides, haemorheology played a crucial role in the brain microcirculation, particularly in acute phase of ischemic stroke. A slightly decrease in blood flow may have influenced on cerebral function greatly. Elevated hemoglobin could not only increase blood viscosity, which affected coronary and cerebral blood flow, but also accelerate the aggregation of red blood cell, leading to platelet aggregation (Atsma et al., 2012;Kalra et al., 2017). In fact, hemoglobin levels have been reported to be positively associated with a variety of cardiovascular diseases, including atherosclerosis and ischemic stroke (Guo et al., 2019). Furthermore, low hemoglobin levels were significantly associated with mortality after AIS (Barlas et al., 2016;Kellert et al., 2011). As for erythrocytes, the changes of oxidative and proteolytic in red blood cell would lead to the cellular rheology, which further influenced inflammatory processes, thus affecting severity of ischemic stroke (Pretorius & Lipinski, 2013). Moreover, anemia and RBC levels had been proposed as predictive indicators in cardiovascular disease and mortality (Ye et al., 2020).
Lymphocyte and platelet played a leading role in inflammation and thrombosis in the pathogenesis of ischemic stroke.

TA B L E 3 The logistic regression analyses of predictors to mortality in 3 months
Lymphocyte, a well-known prognostic element in heart disease, presented a capacity to give rise to acute physiological stress (Li et al., 2015). In many studies, as for subjects of lymphocyte, specific T cells (Macrez et al., 2011) and regulator T cells (Liesz et al., 2009) had a significant impact on eliminating the inflammatory response and regulating the protective function. Moreover, it has already been elucidated that systemic immunosuppression like lymphopenia could be induced by stroke (Yu et al., 2018). In the previous study, cortisol levels could predict short-term outcomes and mortality after AIS (Tu et al., 2013). Lymphopenia could increase the level of pre-stroke baseline cortisol and sympathetic tone (Kim et al., 2012), promoting the production of proinflammatory cytokines that may aggravate ischemic injury (Park et al., 2011) and thus influencing the clinical outcomes. Proved by recent studies, lymphocyte counts of patients died of acute cerebral infarction were lower than that of patients who survived (Gunes & Buyukgol, 2020). Platelet, with a physiological function of hemostasis, was also of crucial importance in acute ischemic stroke. It could not only release various inflammatory mediators, but also develop inflammatory pathways through interacting with cells nearby. In the first place, platelets accumulated where blood vessels were injured, inducing the production of thrombosis and thrombin, which resulted in the start of coagulation process. When platelets were activated, they were used to form atherosclerotic plaques. Via generating thrombosis and stimulating inflammatory reaction, plaques became more stable (Modjeski & Morrell, 2013).

Nevertheless, it was excessive activation and aggregation of
platelets that brought about thrombosis and then caused vascular occlusion, leading to ischemia in cardiovascular and cerebrovascular (Modjeski & Morrell, 2013

TA B L E 4
The cox regression model analyses assessment the death risk