Prognostic value of routine blood tests along with clinical risk factors in predicting ischemic stroke in non-valvular atrial fibrillation: a prospective cohort study

In patients with atrial fibrillation (AF), most biomarkers are still of limited use due to cost-effectiveness and complexity in clinical practice. Biomarkers from routine blood tests improve the current risk stratification in AF patients. This prospective study enrolled 600 patients diagnosed with non-valvular AF, of whom 537 were analyzed. Platelet count; platelet distribution width (PDW); red cell distribution width (RDW); and creatinine, D-dimer, and troponin I levels were measured at enrollment. During the mean follow-up period (2.2 ± 0.6 years), 1.9% patients developed ischemic stroke. According to the optimal cutoff of each biomarker, the risk of ischemic stroke was higher in patients with RDW ≥ 13.5%, creatinine ≥ 1.11 mg/dL, or PDW ≥ 13.2% (significant biomarkers; P value: < 0.01, 0.04, or 0.07, respectively). These 3 significant biomarkers had higher information gain than clinical risk factors in predicting ischemic stroke. The cumulative incidence of ischemic stroke was 1.2%, 1.1%, 8.4%, and 40.0% in patients with 0, 1, 2, and 3 significant biomarkers, respectively (P-for-trend < 0.001). Patients with  ≥ 2 significant biomarkers had a significantly higher risk of ischemic stroke than those with  < 2 significant biomarkers (adjusted hazard ratio 11.5, 95% confidence interval 3.3–40.2, P < 0.001). The predictability for ischemic stroke was significantly improved when  ≥ 2 significant biomarkers were added to the CHA2DS2–VASc score (area under the curve 0.790 vs. 0.620, P = 0.043). Routine blood tests can provide better risk stratification of AF along with clinical risk factors.


Introduction
In the prediction of thromboembolic risk in patients with non-valvular atrial fibrillation (AF), the CHA 2 DS 2 -VASc score (1 point for congestive heart failure, hypertension, age 65-74 years, diabetes mellitus, vascular disease, and female sex, and 2 points for age ≥ 75 years and prior stroke/transient ischemic attack/thromboembolic event) is the most widely used system in clinical practice. However, the CHA 2 DS 2 -VASc score has shown modest predictive ability [1], and thromboembolic events still occur in patients with a CHA 2 DS 2 -VASc score of 0 or 1 [2]. To refine the traditional risk stratification system, approaches based on several biomarkers have emerged for the assessment of thromboembolic risk [3,4]. By employing several biomarkers, new scoring systems have recently been proposed to enhance the predictability of thromboembolic events and to provide guidance for anticoagulation therapy in patients with AF [5,6]. However, in real-world clinical practice, the use of most newly introduced biomarkers is still limited owing to cost-effectiveness issues and complexity of utilization. Further, it is not easy to obtain those biomarkers only for the purpose of risk stratification of patients with nonvalvular AF. Thus, the CHA 2 DS 2 -VASc score remains the most predominant tool for risk stratification in patients with AF despite its limitations. From this viewpoint, we hypothesized that a biomarker would be practically useful if it could be easily obtainable in clinical practice and if it could provide incremental prognostic value over the CHA 2 DS 2 -VASc score. In this regard, we designed a prospective cohort study to identify clinically relevant predictors among easily accessible biomarkers in clinical practice.

Methods
A total of 600 patients who visited the outpatient clinic of Seoul National University Hospital and were diagnosed with non-valvular AF between 2015 and 2017 were prospectively enrolled to estimate the efficacy of simple biomarkers. All patients with AF older than 19 years who agreed to be registered in the study were included. Informed consent was obtained from all patients before enrollment. The exclusion criteria were a history of stroke/transient ischemic attack/thromboembolic events and a prosthetic device exposed to the left heart, such as a left atrial appendage occluder, prosthetic mitral valve, prosthetic aortic valve, and atrial septal defect closing device. Among the initially enrolled 600 patients, 537 patients were included in the final analysis after excluding 10 patients with previous stroke/transient ischemic attack/thromboembolic events not recognized at the initial visit, 24 patients who refused blood sample collection after enrollment, 3 patients who experienced an ischemic stroke event after study enrollment before baseline blood sampling, and 26 patients who were lost to follow-up after the initial visit (Fig. 1). The study protocol was approved by the Institutional Review Board of Seoul National University Hospital (No. 1410-062-619), and this study was conducted in accordance with the Declaration of Helsinki. Informed consent was obtained from all patients before enrollment.
We collected data on biomarkers from all patients within 6 months after enrollment. Platelet count, platelet distribution width (PDW), red cell distribution width (RDW), creatinine level, D-dimer level, and troponin I level were analyzed as markers of platelet reactivity, general patient performance, renal function, thromboembolism, and myocyte injury, respectively. These biomarkers were selected because they are easily obtainable as part of routine blood tests in common practice, and their efficacy in predicting thromboembolic events in patients with AF has been reported [5,[7][8][9][10][11]. To obtain biomarkers, a blood sample was acquired by a professional nurse through vein puncture and was collected in an anticoagulant (ethylenediaminetetraacetic acid [EDTA] or citrate) for CBC test. RDW and PDW were calculated by [standard deviation (SD) of mean corpuscular volume (MCV)/ mean MCV] × 100 for red blood cell and platelet, respectively. Assays for serum creatinine were achieved at the Seoul National University Hospital laboratory. We finally selected the significant biomarkers in predicting the endpoint based on the Cox proportional hazard analysis.
The primary endpoint was thromboembolic events, defined as incident transient ischemic attack, ischemic stroke, or systemic arterial embolism, during the followup period. The follow-up period ended on December 31, 2018. The event duration was defined as the time from the collection of blood samples for evaluating each biomarker to the achievement of the primary endpoint.
Continuous variables are shown as mean ± standard deviation or median (interquartile range) based on the distribution of variables, and categorical variables are shown as number (percentage). Comparisons between 2 groups were performed using the Student's t test or the Mann-Whitney U test for continuous variables, as appropriate. The optimal cutoff of each biomarker was assessed by maximizing the difference in log-rank statistics. Cox proportional hazard regression was performed to examine the risk of the endpoint according to the biomarkers, and biomarkers with P < 0.10 were considered significant biomarkers. The cumulative incidence according to the optimal cutoff of significant biomarkers was compared using Kaplan-Meier survival analysis and the log-rank test. Information gain was used to compare the relative importance of significant biomarkers with clinical risk factors. Variables with higher information gain were considered more important in the prediction of the endpoint. For sensitivity analysis, 10,000 bootstrapping was performed, and the mean value is presented with the 95% confidence interval. The discriminatory ability of the model was estimated using the area under the curve (AUC) in receiver operating characteristic analysis. The reclassification performance of the model with the CHA 2 DS 2 -VASc score and the selected biomarkers was compared with that of model with the CHA 2 DS 2 -VASc score alone, using the category-free net reclassification index (NRI) and the relative integrated discrimination improvement (IDI). P values < 0.05 were considered statistically significant. All analyses were performed using R version 3.5.3 (R Foundation for Statistical Computing, Vienna, Austria).

Results
The baseline characteristics of the study population are shown in Table 1. The mean age was 67.7 ± 7.6 years, and the proportion of patients with a CHA 2 DS 2 -VASc score of ≥ 2 was 66.5%. The proportion of patients who received anticoagulation at enrollment was 71.1%. During the mean follow-up period of 2.2 ± 0.6 years, thromboembolic events occurred in 10 patients and all were ischemic stroke. Patients with incident ischemic stroke were older and had higher CHA 2 DS 2 -VASc scores than those without ischemic stroke. The value of each biomarker at baseline according to the incidence of ischemic stroke is shown in Additional file 1: Supplementary Table 1. According to the optimal cutoff of each biomarker based on maximal log-rank statistics, the risk of ischemic stroke was higher in patients with RDW ≥ 13.5%, creatinine ≥ 1.11 mg/dL, or PDW ≥ 13.2% (P < 0.01, P = 0.04, or P = 0.07, respectively; Table 2). The result was similar after adjustment for anticoagulant use and the CHA 2 DS 2 -VASc score (Table 2). Accordingly, we defined RDW, creatinine, and PDW as significant biomarkers.
The cumulative incidence of ischemic stroke was higher in patients with RDW ≥ 13.5%, creatinine ≥ 1.11 mg/ dL, or PDW ≥ 13.2% than in the other patients (log-rank P < 0.001, P = 0.030, or P = 0.055, respectively; Additional file 2: Supplementary Figure 1). According to the information gain criterion, RDW ≥ 13.5% was the most important predictor of ischemic stroke, followed by creatinine ≥ 1.11 mg/dL and PDW ≥ 13.2%, and all these 3 significant biomarkers had higher information gain than clinical risk factors in the prediction of ischemic stroke (Fig. 2a). The cumulative incidence of ischemic stroke was 1.2%, 1.1%, 8.4%, and 40.0% in patients with 0, 1, 2, and 3 significant biomarkers, respectively (P-for-trend < 0.001, Fig. 2b). The risk of ischemic stroke also increased with an increase in the number of significant biomarkers (Additional file 1: Supplementary Table 2). Patients with ≥ 2 significant biomarkers had a significantly higher risk of ischemic stroke than those with < 2 significant biomarkers (unadjusted hazard ratio [HR] 12.1, 95% CI 3.5-41.9, P < 0.001). This result persisted after adjustment for anticoagulant use and the CHA 2 DS 2 -VASc score (adjusted HR 11.5, 95% CI 3.3-40.2, P < 0.001).

Discussion
In the current prospective study of patients with nonvalvular AF, we aimed to identify clinically relevant biomarkers that are easily accessible in clinical practice and to determine their prognostic value. The major findings were as follows: (1) RDW, creatinine, and PDW were significant biomarkers in predicting ischemic stroke during the prospective follow-up; (2) these 3 biomarkers showed higher importance than clinical risk factors in the prediction of ischemic stroke; (3) the risk of ischemic stroke was significantly higher in patients with ≥ 2 significant biomarkers than in those with < 2 significant biomarkers; (4) the addition of ≥ 2 significant biomarkers to the CHA 2 DS 2 -VASc score provided a significant improvement of the predictive ability for ischemic stroke.
New scoring systems based on biomarkers have been developed and validated. In the subanalysis of a cohort from the ENGAGE AF-TIMI 48 trial (Effective Anticoagulation with Factor Xa Next Generation in AF-Thrombolysis in Myocardial Infarction 48), incorporation of troponin I, N-terminal B-type natriuretic peptide, and D-dimer significantly enhanced the risk assessment for stroke, systemic embolic event, or death in patients with AF [5]. In another subanalysis from the ENGAGE AF-TIMI 48 trial, the thrombolysis in myocardial infarction-AF score was demonstrated to assist in the prediction of a composite clinical outcome [12]. Another novel scoring system-the ABC (age, biomarker, clinical history) risk score, including high-sensitivity troponin T and N-terminal B-type natriuretic peptide-was developed [6], and its performance was recently validated in an external cohort [13]. Although all these scoring systems have  shown the possibility of improving risk stratification in patients with AF by employing biomarkers, their application remains limited. One of the reasons might be the requirement for an additional blood test, time, and cost in real-world clinical practice. In this regard, we focused on biomarkers that can be assessed as part of routine clinical tests without additional blood sampling or cost and sought to comprehensively evaluate their efficacy to improve the current risk stratification of patients with AF. As a result, we found that RDW, creatinine, and PDW were clinically relevant in thromboembolic risk prediction in patients with AF, and that the combination of these factors can provide better predictive ability in addition to clinical risk factors. These 3 biomarkers are robust in terms of generalizability because the values of PDW and RDW are usually available in most patients undergoing routine complete blood count tests, and creatinine is also a general marker usually measured in most patients. Although the exact mechanism should be dissected in the future studies, The prognostic value of each biomarker in patients with AF has already been suggested. RDW has been shown to be a predictor of thromboembolic events or all-cause mortality in patients with AF [14,15]. Consistent with previous studies, we proved the predictive value of RDW for ischemic stroke among patients with AF in a prospective manner. Interestingly, RDW was the most important biomarker in the current study. Several studies seem to support the importance of RDW in patients with AF. An elevated RDW was found to be significantly associated with carotid atherosclerosis [16,17] and was independently correlated with the presence of left atrial/left atrial appendage thrombus [18]. One study suggested that RDW was significantly associated with poor anticoagulation control in patients with AF [19]. Biochemically, RDW has been considered a marker of inflammation, nutritional deficiency, and oxidative stress [20]. These studies implied that RDW represents not a single but several diverse mechanisms of ischemic stroke in patients with AF, which may partially explain the predictive value of RDW was higher than a single risk factor in the current study. The relationship between impaired renal function and increased thromboembolism risk has been well established in patients with AF [21,22]. Its prognostic value for thromboembolic risk and cardiac mortality when added to the CHA 2 DS 2 -VASc score has been proposed [23]. We also demonstrated the efficacy of creatinine in predicting ischemic stroke and postulated that renal function can be considered a marker for enhancing the predictive ability of the CHA 2 DS 2 -VASc score. PDW, a marker reflective of platelet activity, was also a significant biomarker, as in a previous study [7]. One of the determinants of hypercoagulability is platelet hyperactivity, and it is associated with thrombogenesis in AF [24]. Combined together, the 3 significant biomarkers reflect systemic inflammatory status, renal function, and hypercoagulable state, and these attributes may complement the aspect of thromboembolic risk, which cannot be reflected by the CHA 2 DS 2 -VASc score alone.
The current study had several limitations. First, a potential bias might exist because the high-risk group based on the traditional scoring system, not on biomarkers, underwent intensive medical therapy, and this could affect the incidence rate of ischemic stroke. However, we measured the biomarkers at baseline regardless of therapy, and the biomarkers could still reflect the patients' clinical status at enrollment. Second, the cutoff of each biomarker may not be generalizable to different study populations. Despite the limitations, this study still has strengths, as it was a prospective cohort study designed only for the evaluation of simple biomarkers. Third, the number of events was not enough to postulate the linear relationship between significant biomarkers and the incidence of ischemic stroke in the current study. Therefore, future studies are warranted to validate our findings.

Table 2 Identification of significant biomarkers in prediction of incident ischemic stroke
Biomarkers were converted into binary variables based on the optimal cutoff using maximal log-rank statistics CI confidence interval; HR hazard ratio; PDW platelet distribution width; RDW red cell distribution width *Adjusted for anticoagulant use and CHA 2 DS 2 -VASc score † The biomarker with p value < 0.10 was considered as a significant biomarker in prediction of incident ischemic stroke  . 2 a Relative importance of significant biomarkers and clinical risk factors in the prediction of ischemic stroke. b Cumulative incidence of ischemic stroke according to the number of significant biomarkers. The significant biomarkers were RDW ≥ 13.5%, creatinine ≥ 1.11 mg/dL, and PDW ≥ 13.2%. PDW, platelet distribution width; RDW, red cell distribution width Fig. 3 Incremental prognostic value of ≥ 2 significant biomarkers over the CHA 2 DS 2 -VASc score. *category-free NRI. The significant biomarkers were RDW ≥ 13.5%, creatinine ≥ 1.11 mg/dL, and PDW ≥ 13.2%. IDI integrated discrimination improvement; NRI net reclassification index; PDW platelet distribution width; RDW red cell distribution width