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A9) Predicts Delayed Cerebral Ischemia and Outcomes after Aneurysmal Subarachnoid Hemorrhage
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Early Serum Calprotectin (S100A8/A9) Predicts Delayed Cerebral Ischemia and Outcomes after Aneurysmal Subarachnoid Hemorrhage Chenchen Wang, BS,* Yun Kou, MMS,† Yuwei Han, MPharm,* and Xiaoming Li, DSc*
Objective: To investigate the association of early serum calprotectin (S100A8/A9) level with disease severity and prognosis of patients with aneurysmal subarachnoid hemorrhage (aSAH). Patients and Methods: Serum samples were collected from 54 patients with aSAH (within 48 hours of onset) and 54 health controls. Levels of serum calprotectin were determined by enzyme linked immunosorbent assay. The clinical data of aSAH patients were collected. The prognosis was evaluated by modified Rankin scale at 3 months. Univariate and multivariable logistic regression analysis, bivariate correlation analysis and receiver operating characteristic (ROC) curve analysis were used respectively. Results: Serum calprotectin levels were significantly higher in aSAH patients than that in healthy controls (P < .001). The clinical severity was also significantly correlated with the level of serum calprotectin. Patients with poor prognosis at 3 months showed higher serum calprotectin levels within 48 hours of onset than that in patients with good prognosis (P = .002). The level of serum calprotectin within 48 hours was related to the complications of secondary pneumonia. Serum calprotectin can be used as an independent predictor for delayed cerebral ischemia (DCI) after aSAH and poor prognosis in patients with aSAH at 3 months. The ROC curve showed the cutoff value of calprotectin for predicting poor prognosis at 3 months was 6020 pg/ml (sensitivity: 53.57%, specificity: 96.15%), and the cutoff value for predicting DCI was 5275 pg/ml (sensitivity: 68.42%, specificity: 82.86%). Conclusion: Serum calprotectin concentrations within 48 hours after onset was significantly correlated with the clinical severity and the poor prognosis at 3 months in aSAH patients, suggesting that serum calprotectin may be a biomarker for early prediction of prognosis and complications in patients with aSAH and calprotectin may be a target for the treatment of aSAH. Key Words: Aneurysmal subarachnoid hemorrhage—serum calprotectin—poor outcome—delayed cerebral ischemia © 2020 Elsevier Inc. All rights reserved.
Introduction From the *Institute of Neurology, General Hospital of Northern Theater Command, Shenyang, Liaoning, China; and †Special Medicine Departments, General Hospital of Northern Theater Command, Shenyang, Liaoning, China. Received October 12, 2019; revision received January 17, 2020; accepted February 14, 2020. Financial disclosure: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Address correspondence to Xiaoming Li, PhD, Institute of Neurology, General Hospital of Northern Theater Command, No. 83, Wenhua Road, Shenhe District, Shenyang, Liaoning 110016, China. E-mail: [email protected]. 1052-3057/$ - see front matter © 2020 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jstrokecerebrovasdis.2020.104770
Aneurysmal subarachnoid hemorrhage (aSAH) not only has a high early mortality, but also leads to serious long-term neurological dysfunction. Looking for biomarkers that can predict the prognosis of patients will contribute to the decision-making of early treatment. Current clinical scores, such as Glasgow Coma Scale (GCS) score, Hunt and Hess scale, and Fisher grade, can determine the clinical neurological status of patients. However, because of sedation or disturbance of consciousness, the neurological status of patient is sometimes difficult to assess. Biochemical markers can provide specific pathological signals and some information about the severity of
Journal of Stroke and Cerebrovascular Diseases, Vol. &&, No. && (&&), 2020: 104770
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the central nervous system after aSAH. Combined with clinical conditions, these biomarkers can also inform prognosis and guide treatment plan. In the past few years, many biomarkers have been found in blood and cerebrospinal fluid, which may have been reported to predict poor prognosis in patients with aSAH.1-5 However, few markers can apply to clinical applications, more effective markers to predict poor prognosis and complications are still needed. Calprotectin (S100A8/A9, also named myeloid related protein 8/14) is a stable heteromorphic dimer complex of S100A8 and S100A9.6 S100A8 and S100A9 are members of S100s family proteins, they are involved in many human diseases, such as acute and chronic inflammation, tumors, autoimmune diseases, neurodegenerative diseases, and atherosclerosis.7 Extracellular calprotectin is mainly released by activated or necrotic neutrophils and monocytes/macrophages. As an innate immune medium with inflammatory component, they participate in the pathogenesis of various diseases. The level of S100A8/A9 in human peripheral blood is closely related to the number of blood neutrophils. Traditional cardiovascular risk factors such as smoking, obesity, hyperglycemia, and dyslipidemia can increase the expression of S100A8/A9 in peripheral blood. As a potential mediator and prognostic biomarker of coronary artery disease, calprotectin has attracted more and more attention.8,9 Calprotectin participates in the inflammatory cascade and has a wide range of proinflammatory functions, including aggregation of leukocytes, promotion of cytokine and chemokine production, and the regulation of leukocyte adhesion and migration.10 It can be an effective alarmins during many acute and chronic inflammations, such as infections, arthritis, allergies, autoimmune diseases, and other inflammatory diseases.6,11-18 It was reported that compared with unruptured intracranial aneurysms, the gene expression of S100A8 and S100A9 in ruptured intracranial aneurysms was up-regulated19 and serum calprotectin had an immediate increase in patients with acute ischemic stroke.20 And we found an increase in serum calprotectin in patients with aSAH. Up to now, there is no clinical report on the correlation between the expression of serum calprotectin and aSAH. Therefore, the purpose of this study was to investigate whether serum level of calprotectin is associated with clinical severity, complications, and the applicability of serum calprotectin in predicting poor prognosis in aSAH patients.
Materials and Methods Design of Experiment The subjects were hospitalized patients in the Department of Neurosurgery, Shenyang General Military Hospital from December 2016 to January 2018. These patients were diagnosed as aSAH by head computed tomography angiography and further digital subtraction angiography. The
characteristics, treatment, complications, and prognosis of these patients were recorded in our database and analyzed retrospectively. All patients included in the study were admitted to hospital within 48 hours of onset of aSAH and subsequently underwent surgical treatment. Patients with the following conditions were excluded from the study, including those who refused surgery, data incomplete, as well as other systemic diseases, such as autoimmune diseases and malignancies. The serum samples of healthy controls were collected from the health examination center, in which people with colds, acute and chronic inflammation, and systemic diseases were excluded. This analysis is a secondary use of information previously collected during normal hospitalization. The hospital ethics committee approved the study. In accordance with the guidelines of the Helsinki Declaration, patients or their families signed a letter of informed participation.
Clinical Parameters Our database collects basic information about the patient's gender, age, and past medical history. Neurologic assessment was performed through the GCS score21 and Hunt-Hess scale.22 The severity of aSAH was assessed by Fisher' grade determined23 by head CT at admission. Information such as the location, shape, and size of the aneurysm were determined by imaging studies. All patients were followed up by telephone enquiry at 3 months after discharge, and modified Rankin scale (mRS) was used as the prognostic score. The mRS score of 3-6 was classified as poor prognosis (including death patients), and mRS score of 0-2 was classified as good prognosis. The diagnosis of delayed cerebral ischemia (DCI) level was based on “The occurrence of focal neurological impairment (such as hemiparesis, aphasia, apraxia, hemianopia, or neglect), or a decrease of at least 2 points on the GCS (either on the total score or on one of its individual components [eye, motor on either side, verbal]). This should last for at least 1 hour, is not apparent immediately after aneurysm occlusion, and cannot be attributed to other causes by means of clinical assessment, CT or MRI scanning of the brain, and appropriate laboratory studies”.24
Sample Collection and Analysis Peripheral venous blood samples were collected from all patients within 1 hour after admission. The serum samples of the healthy control group were collected during their normal physical examination. After centrifugation at 3000 rpm 4°C for 10 minutes, all serum was separated and stored at 75°C until assayed. enzyme linked immunosorbent assay kit (Shanghai enzyme linked Biotechnology Co., Ltd.) was used to determine the concentration of serum calprotectin according to the manufacturer’s protocol. Each serum sample was repeated 3 times for parallel detection.
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Statistical Analysis Categorical variables were reported as count and proportions in each group, and the continuous data were presented as medians with interquartile ranges (IQRs). Normality of data distribution was tested using the Kolmogorov-Smirnov test or Shapiro-Wilk test. Mann-Whitney U test analysis was used for comparisons between groups for continuous variables. Bivariate correlations were analyzed using Spearman’s correlation coefficient. To confirm independent predictors of poor prognosis of 3 month, if the variables were shown to be significant in the univariate analysis, the variables were included in the multivariate logistic model. Linear regression was used to test the collinearity of variables. Odds ratio (OR) and 95% confidence interval (CI) were calculated. P value less than 0.05 was a significant difference. The receiver operating characteristic (ROC) curve was used to calculate the predictive power of the variable. Estimate the area under the curve (AUC) and the corresponding 95% CI value. Statistical analysis was performed using SPSS version 18.0, MedCalc 19.0.6 and GraphPad Prism 5.
Results Clinical Information of Patients A total of 54 patients with aSAH were included in the study, including 31 women and 23 men, with a median age of 58.50 (IQR 50.75-63.25) years. The healthy control group consisted of 32 females and 22 males with a median age of 59.50 (IQR 47.00-65.25) years old. The intergroup differences in the age (P = .572) and sex (P = .845) were not statistically significant. These selected patients were scored according to different scoring methods. According to the Hunt-Hess scale, 11 (20.4%) patients were Grade I, 21 (38.9%) were Grade II, and there were 11 (20.4%) patients in Grade III, and 11 (20.4%) in Grade IV, respectively. According to Fisher score, 8 (14.8%) patients were Grade I, 18 (33.3%) patients were Grade II, and 9 (16.7%) patients were Grade IV. Of the 54 patients with aneurysms, 29 were posterior circulation aneurysms and 25 were anterior circulation aneurysms (Table 1).
Correlation between Serum Calprotectin Levels and Clinical Indexes To understand the correlation between serum calprotectin levels and clinical indexes of patients, we used bivariate correlation analysis to analyze the correlation between the serum calprotectin level and other indexes (Table 2). We found that serum calprotectin concentrations were significantly correlated with GCS score, Fisher grade, Hunt-Hess scale, and serum C-reactive protein (CRP) levels, respectively (Table 2). Serum calprotectin level was negatively correlated with GCS score at admission (r = 0.300, P = .029) (Fig 1A). Besides, it was positively correlated with
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Table 1. Basic information of aSAH patients and healthy controls Frequency (percentage)/ median (interquartile range) Controls, n Age (years), Gender (female) aSAH, n Age (years) Gender (female) Pneumonia Blood-collecting time Treatment modality Neurosurgical clipping, Endovascular coiling Hunt and Hess grade Grade I Grade II Grade III Grade IV Grade V Fischer grade Grade I Grade II Grade III Grade IV Aneurysm location Anterior circulation Posterior circulation mRS 0-2 3-6 Laboratory findings CRP, mg/L Calprotectin, ng/mL D-dimer, mg/L FEU Hematocrit, % Platelets, 109/L Hemoglobin, g/L Albumin, g/L Bilirubin, mmol/L Sodium, mmol/L Potassium, mmol/L Glucose, mmol/L Fibrinogen, g/L PT, s
54 59.50 (47.00-65.25) 32 (59.3%) 54 58.50 (50.75-63.25) 31 (57.4%) 35 (64.8%) 18 (13-28.25) 25 (46.3%) 29 (53.7%) 11 (20.4%) 21 (38.9%) 11 (20.4%) 11 (20.4%) 0 (0%) 8 (14.8%) 18 (33.3%) 19 (35.2%) 9 (16.7%) 15 (46.3%) 29 (53.7%) 26 (48.1%) 28 (51.9%) 19.20 (10.55-43.90) 4.28 (2.69-7.24) 1.82 (0.87-3.21) 42.00 (40.33-45.88) 234.50 (197.25-273.00) 137.00 (127.00-147.75) 43.60 (40.50-46.70) 11.10 (8.75-15.65) 137.50 (135.75-139.50) 3.77 (3.58-4.02) 8.19 (6.82-10.12) 3.03 (2.67-3.81) 13.10 (12.60-13.50)
CRP concentrations (r = 0.314, P = .021; Fig 1B), Fisher grade (r = 0.425, P = .001; Fig 1C), and Hunt-Hess grade (r = 0.325, P = .009; Fig 1D), respectively.
Serum Calprotectin Levels after aSAH First, we compared serum calprotectin levels in 54 aSAH patients and 54 healthy controls. In the aSAH group, the median calprotectin level was 4284.90 (IQR 2699.91-7241.66) pg/ml, while the healthy controls group was 837.51 (IQR 369.28-1985.69) pg/ml. Serum
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Table 2. The factors correlated with serum calprotectin levels among aSAH patients r value Age (years) Gender (female) Treatment modality (Neurosurgical clipping/endovascular coiling) GCS Hunt and Hess grade Fischer grade Aneurysm location (Anterior circulation/posterior circulation CRP, mg/L D-dimer Hematocrit, % Platelets, 109/L Hemoglobin, g/L Albumin, g/L Bilirubin, mmol/L Sodium, mmol/L Potassium, mmol/L Glucose, mmol/L Fibrinogen, g/L PT
P value
0.146 0.087 0.156
.249 .534 .260
0.300 0.352 0.366 0.067
.029* .009** .007** .628
0.314 0.131 0.051 0.091 0.031 0.189 0.079 0.028 0.068 0.020 0.131 0.001
.021* .350 .720 .520 .827 .184 .572 .845 .630 .888 .351 .995
*P < 0.05. **P < 0.01.
calprotectin levels were significantly higher in aSAH patients than in healthy controls (P < .001; Fig 2A). Next, according to the time of admission, patients were divided into 4 groups (ie, <12 hours, 12-24 hours, 2436 hours, and 36-48 hours), and serum calprotectin levels
were calculated for each group. Because all blood samples were collected within 1 hour after admission, this data also represents serum calprotectin levels at different time points after the onset of aSAH. The results showed that serum calprotectin levels increased significantly within 12 hours after onset, reached the peak at about 2436 hours, and decreased again after 36-48 hours (Fig 2A).
Relationship between Serum Calprotectin Levels and Complications of Pneumonia Complications of secondary pneumonia after SAH usually occur 48 hours after the onset or later. According to the medical records of the 54 patients, 35 patients developed pneumonia 72 hours after admission, and 19 patients did not develop pneumonia. We would like to prove if there was a relationship between serum calprotectin levels and pneumonia complications. We further divided 54 patients into a pneumonia group and a nonpneumonia group, and retrospectively compared the serum calprotectin levels between the 2 groups (the serum samples were the first blood sample of blood drawn at admission). The results showed that the serum calprotectin levels of patients with secondary pneumonia complications were significantly higher than that without pneumonia (median 5197.22 (IQR 3315.09-8560.17) versus median 3516.94 (IQR 2623.534509.64) pg/ml, P = .019; Fig 2B). And this result suggests that patients with high serum levels are more prone to pneumonia complications. According to univariate logistic regression analysis, as shown in the Table 3, all 3 factors of GCS score, Fisher
Figure 1. Correlation between serum calprotectin levels and clinical severity.
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Figure 2. Level of serum calprotectin concentrations in aSAH patients.
grade and serum calprotectin are related to the occurrence of pneumonia. For serum calprotectin, when serum level of calprotectin increased by 1 ng/ml, the unadjusted risk of pneumonia increased by 33.3% (OR, 1.333; 95% CI, 1.038-1.712; P < .001). But by the multivariate analysis, serum calprotectin cannot be used as an independent predictor of pneumonia (Table 2).
Relevance between Serum Calprotectin Level and Occurrence of DCI in aSAH Patients DCI is a common and serious complication after aSAH, which is related to the increase of disability and mortality. According to the DCI criteria, 19 of 54 patients developed DCI and 39 did not develop DCI. The serum calprotectin levels of these 19 people were compared with those of 39
Table 3. Univariate and multivariate logistic regression analysis of pneumonia after aneurysmal subarachnoid hemorrhage Univariate analysis
Age Gender GCS Hunt-Hess Fisher CRP, mg/L Calprotectin, ng/mL D-dimer, mg/L FEU Hematocrit, % Platelets, 109/L Hemoglobin, g/L Albumin, g/L Bilirubin, mmol/L Sodium, mmol/L Potassium, mmol/L Glucose, mmol/L Fibrinogen, g/L PT, s
Multivariate analysis
OR (95% CI)
P value
0.983 (0.929-1.041) 0.970 (0.313-3.003) 0.774 (0.629-0.963) 1.745 (0.958-3.179) 3.510 (1.579-7.801) 1.020 (0.993-1.048) 1.333 (1.038-1.712) 1.027 (0.892-1.182) 0.957 (0.884-1.037) 1.005 (0.995-1.015) 0.993 (0.966-1.022) 0.963 (0.850-1.091) 1.005 (0.884-1.142) 1.161 (0.960-1.403) 0.985 (0.257-3.778) 1.223 (0.921-1.623) 0.783 (0.393-1.561) 0.735 (0.334-1.620)
.559 .970 .016 .069 .002 .148 .024 .710 .283 .355 .640 .556 .941 .124 .982 .164 .488 .446
OR (95% CI)
P value
0.893 (0.684-1.165)
.404
2.194 (0810-5.946)
.122
1.211 (0.915-2.647)
.180
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Table 4. Univariate and multivariate logistic regression analysis of DCI after aneurysmal subarachnoid hemorrhage Univariate analysis
Age Gender GCS Hunt-Hess Fisher CRP, mg/L Calprotectin, ng/mL D-dimer, mg/L FEU Hematocrit, % Platelets, 109/L Hemoglobin, g/L Albumin, g/L Bilirubin, mmol/L Sodium, mmol/L Potassium, mmol/L Glucose, mmol/L Fibrinogen, g/L PT, s
Multivariate analysis
OR (95% CI)
P value
1.003 (0.948-1.061) 0.489 (0.151-1.579) 0.706 (0.575-0.865) 2.353 (1.262-4.390) 3.339 (1.517-7.350) 1.027 (1.003-1.053) 1.517 (1.181-1.948) 1.049 (0.922-1.194) 0.965 (0.880-1.057) 0.997 (0.987-1.006) 1.005 (0.977-1.033) 1.138 (0.983-1.318) 0.960 (0.880-1.047) 0.940 (0.783-1.129) 0.597 (0.157-2.266) 1.143 (0.889-1.471) 0.761 (0.374-1546) 0.576 (0.241-1.375)
0.920 0.231 0.001 0.007 0.003 0.030 0.001 0.467 0.441 0.486 0.731 0.082 0.353 0.509 0.448 0.297 0.450 0.214
OR (95% CI)
P value
0.662 (0.465-0.942) 0.797 (0.300-2.114) 1.154 (0.392-3.392) 1.013 (0.979-1.048) 1.584 (1.112-2.257)
.022* .648 .665 .455 .011*
*P < 0.05.
people, and the results showed that the level of serum calprotectin in patients with DCI was significantly higher than that in patients without DCI (median 6380.89 (IQR 4323.93-11358.94) versus median 3569.67 (IQR 2623.534926.02) pg/ml, P = .001; Fig 2C). In addition, as shown in Table 4, we also conducted a univariate analysis to investigate the independent predictive markers of DCI among the factors. The univariate analysis results showed that among the listed indicators, only GCS score, Fisher grade, Hunt-Hess grade, CRP, and calprotectin were significantly associated with poor prognosis. The analysis shows these variables are not collinear, so we further put them into in the multivariate logistic regression model. The results showed that, like GCS score (OR, 0.662; 95%CI, 0.465-0.942; P = .022), calprotectin (OR, 1.584; 95% CI, 1.112-2.257; P = .011) could be used as an independent predictor of DCI after aSAH. This meant that every 1 ng/ml increase in serum calprotectin as a continuous variable, the risk of adjusted poor outcomes increased by 58.4% and the unadjusted risk increased by 51.7%.
Sensitivity and Specificity of Calprotectin in Predicting DCI after aSAH CRP has been proved to be an effective predictor of SAH25-27 and the early phase CRP levels following SAH is related to delayed ischemia or angiographic vasospasm.28 We compare the sensitivity and specificity of calprotectin with CRP to show the ability of calprotectin to predict DCI after aSAH. The AUC value of calprotectin was 0.782 (95% CI, 0.649-0.883; P < .001; sensitivity: 68.42%,
specificity: 82.86%), while the AUC value of CRP was 0.696 (95% CI, 0.556-0.814; P = .011; sensitivity: 78.95%, specificity: 60.00%; Fig 3A, Table 5). ROC curve suggested that the serum calprotectin level on admission could predict the subsequent DCI, and its predictive value was similar to that of CRP (P = .387). The cutoff value of calprotectin for predicting DCI in patients with aSAH was 5275 pg/ml. Additionally, we also calculated the AUC values of Fisher grade, GCS score and Hunt-Hess scale to predict DCI, respectively (Fig 3B-D). As shown in Table 5, The AUC of GCS, Fisher grade, and Hunt-Hess scale were 0.831 (95% CI, 0.703-0.920; P < .001; sensitivity: 89.47%, specificity: 70.59%), 0.759 (95% CI, 0.624-0.865; P < .001; sensitivity: 78.95%, specificity: 63.86%) and 0.723 (95% CI, 0.585-0.836; P = .012; sensitivity: 68.42%, specificity: 74.29%), respectively. There were no significant differences between the AUC value of serum calprotectin and the AUC value of GCS score, Fisher grade, and Hunt-Hess score (P = .589, P = .821, P = .502, respectively). In the combined logistic regression model, calprotectin did not significantly improve the AUC of these neurological scores (P > .05).
Relevance between Serum Calprotectin Level and Prognosis of aSAH Patients According to the follow-up results of 3 months after discharge, 54 patients were further divided into good prognosis group and poor prognosis group. In our study, 28 patients had a poor prognosis (mRS 3-6) and 26 patients with good prognosis (mRS 0-2). Then the serum calprotectin levels were compared retrospectively between the 2
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Figure 3. Values of variables in predicting delayed cerebral ischemia in patients with aSAH.
groups (Serum samples were the first blood drawn at admission). The data showed that serum calprotectin levels were significantly higher in the poor prognosis group than that in the good prognosis group (median 6364.30 (IQR 4039.90-8930.76) versus median 2920.72 (IQR 2436.664613.73) pg/ml, P < .001), which indicated there were a significant difference between the 2 groups (Fig 2D). To investigate the main factors affecting the prognosis of patients with aSAH, we performed univariate analysis for the indexes listed in Table 6. The univariate analysis results showed that among the listed indicators, only GCS score, Fisher grade, Hunt-Hess grade, CRP, and calprotectin were significantly associated with poor prognosis. In addition, we confirm that these variables are not
collinearity, so they are included in the multivariate logistic regression model. The results showed that only calprotectin (OR, 1.775; 95% CI, 1.190-2.647; P = .005) emerged as an independent predictor of poor prognosis at 3 months. This meant that every 1 ng/ml increase in serum calprotectin as a continuous variable, the risk of adjusted poor outcomes increased by 77.5% and the unadjusted risk increased by 75.2%.
Sensitivity and Specificity of Calprotectin in Predicting 3-Month Prognosis Previous studies have shown that CRP could be used as an effective index to predict the prognosis of SAH.25
Table 5. Receiver operating characteristic curve analysis of factors predicting DCI among aSAH patients
Criterion Area under curve 95% confidence interval Sensitivity 95% confidence interval Specificity 95% confidence interval + likelihood ratio 95% confidence interval likelihood ratio 95% confidence interval P value
Calprotectin
CRP
GCS
Fisher
Hunt-Hess
>5274.66 0.782 0.649-0.883 68.42 43.3-87.4 82.86 66.4-93.4 3.99 1.8-8.8 0.38 0.2-0.8 Reference
>16.2 0.696 0.556-0.814 78.95 54.4-93.9 60.00 42.1-76.1 1.97 1.2-3.2 0.35 0.1-0.9 .387
12 0.831 0.703-0.920 89.47 66.9-98.7 70.59 52.5-84.9 3.04 1.8-5.2 0.15 0.04-0.6 .589
>2 0.759 0.624-0.865 78.95 54.4-93.9 62.86 44.9-78.5 2.13 1.3-3.5 0.33 0.1-0.8 .821
>2 0.723 0.585-0.836 68.42 43.4-87.4 74.29 56.7-87.5 2.66 1.4-5.0 0.43 0.2-0.8 .502
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Table 6. Univariate and multivariate logistic regression analysis of 3-month prognosis after aneurysmal subarachnoid hemorrhage Univariate analysis
Age Female GCS Hunt-Hess Fisher CRP, mg/L Calprotectin, ng/mL D-dimer Hematocrit, % Platelets, 109/L Hemoglobin, g/L Albumin, g/L Bilirubin, mmol/L Sodium, mmol/L Potassium, mmol/L Glucose, mmol/L Fibrinogen, g/L PT
Multivariate analysis
OR (95% CI)
P value
1.019 (0.966-1.076) 0.978 (0.332-2.878) 0.717 (0.584-0.880) 3.211 (1.586-6.500) 3.402 (1.589-7.281) 1.038 (1.007-1.070) 1.752 (1.253-2.541) 1.207 (0.980-1.486) 0.913 (0.807-1.034) 0.997 (0.987-1.006) 0.976 (0.945-1.009) 0.963 (0.850-1.091) 1.024 (0.960-1.091) 1.094 (0.916-1.308) 0.577 (0.151-2.196) 1.038 (0.818-1.319) 1.469 (0.744-2.902) 1.171 (0.547-2.506)
.490 .967 .001 .001 .002 .017 .001 .076 .152 .478 .148 .556 .475 .322 .420 .758 .268 .685
OR (95% CI)
P value
0.826 (0.567-1.203) 1.607 (0.538-4.802) 1.547 (0.452-5.291) 1.011 (0.974-1.050) 1.775 (1.190-2.647)
.320 .396 .487 .558 .005*
*P < 0.05.
Therefore, in this study, we used the ROC curve to measure the ability of serum calprotectin and CRP in predicting the prognosis of patients of aSAH patients with CRP. The AUC values of calprotectin and CRP were 0.809 (95% CI, 0.6790.903; P < .001; sensitivity: 53.57%, specificity: 96.15%), and 0.693 (95% CI, 0.553-0.811; P = .015; sensitivity: 74.29%, specificity: 76.92%), respectively (Fig 4A, Table 7). The ROC
curve showed that serum calprotectin level on admission statistically significantly predicted 3-month poor outcome of patients, and the predictive value of the calprotectin concentration was similar to that of CRP (P = .218). The ROC curve showed serum calprotectin concentrations greater than 6020 pg/ml predicted poor prognosis at 3 months after discharge. In other words, the cutoff value of calprotectin in
Figure 4. Values of variables in predicting poor outcome in patients with aSAH.
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Table 7. Receiver operating characteristic curve analysis of factors predicting 3-month poor outcomes among aSAH patients
Criterion Area under curve 95% confidence interval Sensitivity 95% confidence interval Specificity 95% confidence interval + likelihood ratio 95% confidence interval likelihood ratio 95% confidence interval P value
Calprotectin
CRP
GCS
Fisher
Hunt-Hess
>6.02 0.809 0.679-0.903 53.57 33.9-72.5 96.15 80.4-99.9 13.93 2.0-98.2 0.48 0.3-0.7 Reference
>21.7 0.693 0.553-0.811 64.29 44.1-81.4 76.92 56.4-91.0 2.79 1.3-5.9 0.46 0.3-0.8 .218
22 0.782 0.647-0.884 71.43 51.3-86.8 72.00 50.6-87.9 2.55 1.3-5.0 0.40 0.2-0.7 .747
>2 0.766 0.631-0.870 75.00 55.1-89.3 73.08 52.2-88.4 2.79 1.4-5.4 0.34 0.2-0.7 .543
>2 0.769 0.635-0.873 60.71 40.6-78.5 80.77 60.6-93.4 3.16 1.4-7.3 0.49 0.3-0.8 .626
predicting poor prognosis in patients with aSAH was 6020 pg/ml. In addition, neurological scores are often used to predict prognosis. Here, we calculated the AUC values of Fisher grade, GCS score, and Hunt-Hess scale, respectively in the 54 patients (Fig 4BCD). As shown in Table 5, The AUC of Fisher grade, GCS, and Hunt-Hess scale were 0.766 (95% CI, 0.631-0.870; P = .008; sensitivity:75.00%, specificity:73.08%), 0.782 (95% CI, 0.647-0.984; P = .004; sensitivity:71.43%, specificity:72.00%), and 0.769 (95% CI, 0.635-0.873; P = .007; sensitivity:60.71%, specificity:80.77%). In terms of prognostic predictive ability assessed by AUC, there were no substantial differences between serum calprotectin concentrations and other variables, including GCS score (P = .747), Fisher grade (P = .543), and Hunt-Hess scale (P = .626). In the combined logistic regression model, calprotectin statistically significantly improved the AUC of GCS score (P = .031) and Hunt-Hess scale (P = .021) but did not statistically significantly improve the AUC of Fisher grade (P = .073).
Discussion The correlation between serum concentrations of calprotectin (S100A8/A9) and inflammation has been well described. The S100A8/A9 complex has been proved to be a diagnostic marker of inflammation, especially in noninfectious inflammatory diseases such as arthritis, chronic inflammatory lung disease, and intestinal disease.29 Calprotectin mainly exists in the cytoplasm of inflammatory cells such as granulocytes and monocytes. In the central nervous system, calprotectin is abundantly expressed in microvascular endothelial cells and microglial cells.30 However, there is no report on the relevance between calprotectin and aSAH. We firstly reported that serum calprotectin levels were significantly elevated in aSAH patients within 48 hours after onset compared with the healthy controls. According to our statistical results, calprotectin levels increased significantly within 12 hours after onset, peaked at approximately 24-36 hours, and then decreased again between 36 and 48 hours. This
phenomenon indicates that calprotectin is a marker of acute inflammatory response in SAH patients. Further retrospective statistical analysis showed that patients with subsequent pneumonia complications had significantly higher serum calprotectin levels on admission than patients without pneumonia complications, but in the multivariate logistics analysis, serum calprotectin cannot be used as an independent predictor of pneumonia. However, the statistical results showed that, like GCS score and Fisher grade, serum calprotectin was related to the occurrence of pneumonia, which suggests that calprotectin is an early marker of inflammation in SAH patients and it is associated with the complications of secondary pneumonia. This may be related to the role of calcitonin in the pathogenesis of pneumonia. It was reported that high level of S100A8 and S100A9 in the alveolar walls of lungs of mice infected with S. pneumoniae involved in the transepithelial migration of macrophages and neutrophils31 and calprotectin has a strong link between local alveolar inflammation, neutrophil activation, and NETosis.32 Moreover, it has been reported that calprotectin is an endogenous ligand of toll-like receptor 4 (TLR4) and of the receptor for advanced glycation end products (RAGE)33 .Calprotectin also activates the MAPK and NFkB signaling pathways, which leads to the amplification of the proinflammatory cascade through TLR4 and RAGE pathways.34 Coincidentally, in the early stage of SAH, the main activation of signaling pathway is TLR4-related pathway,35 and RAGE is also directly involved in the inflammatory response after SAH.36 Based on these studies, we speculate that as an endogenous ligand for TLR4 and RAGE, calprotectin significantly increased in the early stage of SAH and bind to TLR4 and RAGE, thus participating in the activation of TLR4 and RAGE signal transduction pathways. Recent studies have shown that DCI after aSAH is associated with vasospasm, microcirculation disorder and microthrombosis, cortical diffusion inhibition, and inflammation. In our study, analyzed by multivariate logistic regression, the result showed that calprotectin can be used as an
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independent predictor of DCI emergence. Moreover, the results of ROC curve showed that the predictive ability of calprotectin to DCI was similar to that of other standard clinical scoring systems such as Fisher grade, GCS score, Hunt-Hess score, and similar to that of CRP. The exact mechanism by which calprotectin is involved in DCI is unclear, but it has been reported that the calprotectin can bind to arachidonic acid (AA), the calprotectin-AA complex promotes the transcellular transport of AA, which leads to vascular endothelial injury and vascular inflammation.37,38 In addition, the transfer of AA can promote the activation of NADPH oxidase to produce ROS in phagocytes.39 Our statistical results showed that, by multivariate logistic regression model, among the factors associated with 3-month poor prognosis of aSAH, only the calprotectin is an independent predictor of poor prognosis at 3 months (P = .005). There may be many mechanisms by which calprotectin affects prognosis including consciousness level and limb motor function in patients with SAH, but we think there are 2 most important points, 1 of which is cerebral vasospasm and another mechanism is neuronal cell death. The onset of aSAH elicits activation of the inflammatory cascade, and ongoing neuroinflammation leads to secondary complications, such as vasospasm, DCI, and neuronal cell death. And these secondary complications have been considered to be the causes of neurocognitive impairment.1 Calprotectin is involved in the inflammatory cascade and has a wide range of proinflammation effects, therefore high serum calprotectin correlates with poor outcome. Furthermore, our data analysis showed that the calprotectin has a good accuracy in predicting adverse outcome. The AUC of the calprotectin concentration was similar to those of CRP, Fisher grade, GCS score, and Hunt-Hess scale for the prediction of these poor outcomes, while in the combined logistic-regression models, calprotectin improved the predict ability of clinical scores. These further analyses all revealed that serum level of calprotectin was independently associated with poor outcome and suggested that calprotectin can be a good predictor associated with severity and prognosis of aSAH. In fact, calprotectin has been studied as a biomarker in several diseases. Calprotectin has been found to be associated with the progression of inflammatory bowel disease. And fecal calprotectin can predict and monitor the risk of recurrence in patients with inflammatory bowel disease after treatment.40 Besides, calprotectin has been found to play an important role in the rheumatoid arthritis, and plasma calprotectin has the strongest correlation with assessments of disease activity.11 In patients undergoing cardiac surgery, plasma calprotectin was significantly increased, and can predict the onset of acute kidney injury.41 S100A9 knockout in mice reduces TBI-induced neuroinflammation, protect the brain from secondary damage by TBI, and improves functional outcome after TBI.42
We propose for the first time that the level of serum calprotectin can be used as an independent predictor of DCI after aSAH and the prognosis of aSAH and serum calprotectin is not only an objective indicator, but also a new and effective target for the treatment of aSAH. Anticalprotectin antibodies and their antagonists may be used in the treatment of SAH. However, our current study still has some limitations. First, we did not continue to track the changes in calprotectin over a longer period. We are limited to talking about acute inflammatory cascade of aSAH. However, if peripheral blood and cerebrospinal fluid can be obtained from the same patients at different time points, serial change of calprotectin concentrations can be studied. Second, we are limited to talking about 3month prognosis. If there were longer follow-up records, we would be able to discuss the relationship between aSAH mortality rate and calprotectin. And our conclusions still need to be validated in a larger population of multicenter. Last but not least, the cause of elevated serum in patients with aSAH and its mechanism in the pathology of SAH should be further in-depth studied.
Conclusion In conclusion, serum calprotectin level within 48 hours after onset in patients with aSAH has high predictive value of prognosis and complications. Serum calprotectin could be used as an independent predictor of poor prognosis at 3 months as well as DCI emergence. Serum calprotectin level was related to the occurrence of secondary pneumonia complications after aSAH but cannot be used an independent predictor of secondary pneumonia. Therefore, it may become a prognostic biomarker of aSAH. Declaration of Interest: financial interests.
The authors declare no competing
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Early Serum Calprotectin (S100A8/A9) Predicts Delayed Cerebral Ischemia and Outcomes after Aneurysmal Subarachnoid Hemorrhage Chenchen Wang, BS,* Yun Kou, MMS,† Yuwei Han, MPharm,* and Xiaoming Li, DSc*
Objective: To investigate the association of early serum calprotectin (S100A8/A9) level with disease severity and prognosis of patients with aneurysmal subarachnoid hemorrhage (aSAH). Patients and Methods: Serum samples were collected from 54 patients with aSAH (within 48 hours of onset) and 54 health controls. Levels of serum calprotectin were determined by enzyme linked immunosorbent assay. The clinical data of aSAH patients were collected. The prognosis was evaluated by modified Rankin scale at 3 months. Univariate and multivariable logistic regression analysis, bivariate correlation analysis and receiver operating characteristic (ROC) curve analysis were used respectively. Results: Serum calprotectin levels were significantly higher in aSAH patients than that in healthy controls (P < .001). The clinical severity was also significantly correlated with the level of serum calprotectin. Patients with poor prognosis at 3 months showed higher serum calprotectin levels within 48 hours of onset than that in patients with good prognosis (P = .002). The level of serum calprotectin within 48 hours was related to the complications of secondary pneumonia. Serum calprotectin can be used as an independent predictor for delayed cerebral ischemia (DCI) after aSAH and poor prognosis in patients with aSAH at 3 months. The ROC curve showed the cutoff value of calprotectin for predicting poor prognosis at 3 months was 6020 pg/ml (sensitivity: 53.57%, specificity: 96.15%), and the cutoff value for predicting DCI was 5275 pg/ml (sensitivity: 68.42%, specificity: 82.86%). Conclusion: Serum calprotectin concentrations within 48 hours after onset was significantly correlated with the clinical severity and the poor prognosis at 3 months in aSAH patients, suggesting that serum calprotectin may be a biomarker for early prediction of prognosis and complications in patients with aSAH and calprotectin may be a target for the treatment of aSAH. Key Words: Aneurysmal subarachnoid hemorrhage—serum calprotectin—poor outcome—delayed cerebral ischemia © 2020 Elsevier Inc. All rights reserved.
Introduction From the *Institute of Neurology, General Hospital of Northern Theater Command, Shenyang, Liaoning, China; and †Special Medicine Departments, General Hospital of Northern Theater Command, Shenyang, Liaoning, China. Received October 12, 2019; revision received January 17, 2020; accepted February 14, 2020. Financial disclosure: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Address correspondence to Xiaoming Li, PhD, Institute of Neurology, General Hospital of Northern Theater Command, No. 83, Wenhua Road, Shenhe District, Shenyang, Liaoning 110016, China. E-mail: [email protected]. 1052-3057/$ - see front matter © 2020 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jstrokecerebrovasdis.2020.104770
Aneurysmal subarachnoid hemorrhage (aSAH) not only has a high early mortality, but also leads to serious long-term neurological dysfunction. Looking for biomarkers that can predict the prognosis of patients will contribute to the decision-making of early treatment. Current clinical scores, such as Glasgow Coma Scale (GCS) score, Hunt and Hess scale, and Fisher grade, can determine the clinical neurological status of patients. However, because of sedation or disturbance of consciousness, the neurological status of patient is sometimes difficult to assess. Biochemical markers can provide specific pathological signals and some information about the severity of
Journal of Stroke and Cerebrovascular Diseases, Vol. &&, No. && (&&), 2020: 104770
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the central nervous system after aSAH. Combined with clinical conditions, these biomarkers can also inform prognosis and guide treatment plan. In the past few years, many biomarkers have been found in blood and cerebrospinal fluid, which may have been reported to predict poor prognosis in patients with aSAH.1-5 However, few markers can apply to clinical applications, more effective markers to predict poor prognosis and complications are still needed. Calprotectin (S100A8/A9, also named myeloid related protein 8/14) is a stable heteromorphic dimer complex of S100A8 and S100A9.6 S100A8 and S100A9 are members of S100s family proteins, they are involved in many human diseases, such as acute and chronic inflammation, tumors, autoimmune diseases, neurodegenerative diseases, and atherosclerosis.7 Extracellular calprotectin is mainly released by activated or necrotic neutrophils and monocytes/macrophages. As an innate immune medium with inflammatory component, they participate in the pathogenesis of various diseases. The level of S100A8/A9 in human peripheral blood is closely related to the number of blood neutrophils. Traditional cardiovascular risk factors such as smoking, obesity, hyperglycemia, and dyslipidemia can increase the expression of S100A8/A9 in peripheral blood. As a potential mediator and prognostic biomarker of coronary artery disease, calprotectin has attracted more and more attention.8,9 Calprotectin participates in the inflammatory cascade and has a wide range of proinflammatory functions, including aggregation of leukocytes, promotion of cytokine and chemokine production, and the regulation of leukocyte adhesion and migration.10 It can be an effective alarmins during many acute and chronic inflammations, such as infections, arthritis, allergies, autoimmune diseases, and other inflammatory diseases.6,11-18 It was reported that compared with unruptured intracranial aneurysms, the gene expression of S100A8 and S100A9 in ruptured intracranial aneurysms was up-regulated19 and serum calprotectin had an immediate increase in patients with acute ischemic stroke.20 And we found an increase in serum calprotectin in patients with aSAH. Up to now, there is no clinical report on the correlation between the expression of serum calprotectin and aSAH. Therefore, the purpose of this study was to investigate whether serum level of calprotectin is associated with clinical severity, complications, and the applicability of serum calprotectin in predicting poor prognosis in aSAH patients.
Materials and Methods Design of Experiment The subjects were hospitalized patients in the Department of Neurosurgery, Shenyang General Military Hospital from December 2016 to January 2018. These patients were diagnosed as aSAH by head computed tomography angiography and further digital subtraction angiography. The
characteristics, treatment, complications, and prognosis of these patients were recorded in our database and analyzed retrospectively. All patients included in the study were admitted to hospital within 48 hours of onset of aSAH and subsequently underwent surgical treatment. Patients with the following conditions were excluded from the study, including those who refused surgery, data incomplete, as well as other systemic diseases, such as autoimmune diseases and malignancies. The serum samples of healthy controls were collected from the health examination center, in which people with colds, acute and chronic inflammation, and systemic diseases were excluded. This analysis is a secondary use of information previously collected during normal hospitalization. The hospital ethics committee approved the study. In accordance with the guidelines of the Helsinki Declaration, patients or their families signed a letter of informed participation.
Clinical Parameters Our database collects basic information about the patient's gender, age, and past medical history. Neurologic assessment was performed through the GCS score21 and Hunt-Hess scale.22 The severity of aSAH was assessed by Fisher' grade determined23 by head CT at admission. Information such as the location, shape, and size of the aneurysm were determined by imaging studies. All patients were followed up by telephone enquiry at 3 months after discharge, and modified Rankin scale (mRS) was used as the prognostic score. The mRS score of 3-6 was classified as poor prognosis (including death patients), and mRS score of 0-2 was classified as good prognosis. The diagnosis of delayed cerebral ischemia (DCI) level was based on “The occurrence of focal neurological impairment (such as hemiparesis, aphasia, apraxia, hemianopia, or neglect), or a decrease of at least 2 points on the GCS (either on the total score or on one of its individual components [eye, motor on either side, verbal]). This should last for at least 1 hour, is not apparent immediately after aneurysm occlusion, and cannot be attributed to other causes by means of clinical assessment, CT or MRI scanning of the brain, and appropriate laboratory studies”.24
Sample Collection and Analysis Peripheral venous blood samples were collected from all patients within 1 hour after admission. The serum samples of the healthy control group were collected during their normal physical examination. After centrifugation at 3000 rpm 4°C for 10 minutes, all serum was separated and stored at 75°C until assayed. enzyme linked immunosorbent assay kit (Shanghai enzyme linked Biotechnology Co., Ltd.) was used to determine the concentration of serum calprotectin according to the manufacturer’s protocol. Each serum sample was repeated 3 times for parallel detection.
ARTICLE IN PRESS CEREBRAL ISCHEMIA AND OUTCOMES AFTER ANEURYSMAL SUBARACHNOID HEMORRHAGE
Statistical Analysis Categorical variables were reported as count and proportions in each group, and the continuous data were presented as medians with interquartile ranges (IQRs). Normality of data distribution was tested using the Kolmogorov-Smirnov test or Shapiro-Wilk test. Mann-Whitney U test analysis was used for comparisons between groups for continuous variables. Bivariate correlations were analyzed using Spearman’s correlation coefficient. To confirm independent predictors of poor prognosis of 3 month, if the variables were shown to be significant in the univariate analysis, the variables were included in the multivariate logistic model. Linear regression was used to test the collinearity of variables. Odds ratio (OR) and 95% confidence interval (CI) were calculated. P value less than 0.05 was a significant difference. The receiver operating characteristic (ROC) curve was used to calculate the predictive power of the variable. Estimate the area under the curve (AUC) and the corresponding 95% CI value. Statistical analysis was performed using SPSS version 18.0, MedCalc 19.0.6 and GraphPad Prism 5.
Results Clinical Information of Patients A total of 54 patients with aSAH were included in the study, including 31 women and 23 men, with a median age of 58.50 (IQR 50.75-63.25) years. The healthy control group consisted of 32 females and 22 males with a median age of 59.50 (IQR 47.00-65.25) years old. The intergroup differences in the age (P = .572) and sex (P = .845) were not statistically significant. These selected patients were scored according to different scoring methods. According to the Hunt-Hess scale, 11 (20.4%) patients were Grade I, 21 (38.9%) were Grade II, and there were 11 (20.4%) patients in Grade III, and 11 (20.4%) in Grade IV, respectively. According to Fisher score, 8 (14.8%) patients were Grade I, 18 (33.3%) patients were Grade II, and 9 (16.7%) patients were Grade IV. Of the 54 patients with aneurysms, 29 were posterior circulation aneurysms and 25 were anterior circulation aneurysms (Table 1).
Correlation between Serum Calprotectin Levels and Clinical Indexes To understand the correlation between serum calprotectin levels and clinical indexes of patients, we used bivariate correlation analysis to analyze the correlation between the serum calprotectin level and other indexes (Table 2). We found that serum calprotectin concentrations were significantly correlated with GCS score, Fisher grade, Hunt-Hess scale, and serum C-reactive protein (CRP) levels, respectively (Table 2). Serum calprotectin level was negatively correlated with GCS score at admission (r = 0.300, P = .029) (Fig 1A). Besides, it was positively correlated with
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Table 1. Basic information of aSAH patients and healthy controls Frequency (percentage)/ median (interquartile range) Controls, n Age (years), Gender (female) aSAH, n Age (years) Gender (female) Pneumonia Blood-collecting time Treatment modality Neurosurgical clipping, Endovascular coiling Hunt and Hess grade Grade I Grade II Grade III Grade IV Grade V Fischer grade Grade I Grade II Grade III Grade IV Aneurysm location Anterior circulation Posterior circulation mRS 0-2 3-6 Laboratory findings CRP, mg/L Calprotectin, ng/mL D-dimer, mg/L FEU Hematocrit, % Platelets, 109/L Hemoglobin, g/L Albumin, g/L Bilirubin, mmol/L Sodium, mmol/L Potassium, mmol/L Glucose, mmol/L Fibrinogen, g/L PT, s
54 59.50 (47.00-65.25) 32 (59.3%) 54 58.50 (50.75-63.25) 31 (57.4%) 35 (64.8%) 18 (13-28.25) 25 (46.3%) 29 (53.7%) 11 (20.4%) 21 (38.9%) 11 (20.4%) 11 (20.4%) 0 (0%) 8 (14.8%) 18 (33.3%) 19 (35.2%) 9 (16.7%) 15 (46.3%) 29 (53.7%) 26 (48.1%) 28 (51.9%) 19.20 (10.55-43.90) 4.28 (2.69-7.24) 1.82 (0.87-3.21) 42.00 (40.33-45.88) 234.50 (197.25-273.00) 137.00 (127.00-147.75) 43.60 (40.50-46.70) 11.10 (8.75-15.65) 137.50 (135.75-139.50) 3.77 (3.58-4.02) 8.19 (6.82-10.12) 3.03 (2.67-3.81) 13.10 (12.60-13.50)
CRP concentrations (r = 0.314, P = .021; Fig 1B), Fisher grade (r = 0.425, P = .001; Fig 1C), and Hunt-Hess grade (r = 0.325, P = .009; Fig 1D), respectively.
Serum Calprotectin Levels after aSAH First, we compared serum calprotectin levels in 54 aSAH patients and 54 healthy controls. In the aSAH group, the median calprotectin level was 4284.90 (IQR 2699.91-7241.66) pg/ml, while the healthy controls group was 837.51 (IQR 369.28-1985.69) pg/ml. Serum
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Table 2. The factors correlated with serum calprotectin levels among aSAH patients r value Age (years) Gender (female) Treatment modality (Neurosurgical clipping/endovascular coiling) GCS Hunt and Hess grade Fischer grade Aneurysm location (Anterior circulation/posterior circulation CRP, mg/L D-dimer Hematocrit, % Platelets, 109/L Hemoglobin, g/L Albumin, g/L Bilirubin, mmol/L Sodium, mmol/L Potassium, mmol/L Glucose, mmol/L Fibrinogen, g/L PT
P value
0.146 0.087 0.156
.249 .534 .260
0.300 0.352 0.366 0.067
.029* .009** .007** .628
0.314 0.131 0.051 0.091 0.031 0.189 0.079 0.028 0.068 0.020 0.131 0.001
.021* .350 .720 .520 .827 .184 .572 .845 .630 .888 .351 .995
*P < 0.05. **P < 0.01.
calprotectin levels were significantly higher in aSAH patients than in healthy controls (P < .001; Fig 2A). Next, according to the time of admission, patients were divided into 4 groups (ie, <12 hours, 12-24 hours, 2436 hours, and 36-48 hours), and serum calprotectin levels
were calculated for each group. Because all blood samples were collected within 1 hour after admission, this data also represents serum calprotectin levels at different time points after the onset of aSAH. The results showed that serum calprotectin levels increased significantly within 12 hours after onset, reached the peak at about 2436 hours, and decreased again after 36-48 hours (Fig 2A).
Relationship between Serum Calprotectin Levels and Complications of Pneumonia Complications of secondary pneumonia after SAH usually occur 48 hours after the onset or later. According to the medical records of the 54 patients, 35 patients developed pneumonia 72 hours after admission, and 19 patients did not develop pneumonia. We would like to prove if there was a relationship between serum calprotectin levels and pneumonia complications. We further divided 54 patients into a pneumonia group and a nonpneumonia group, and retrospectively compared the serum calprotectin levels between the 2 groups (the serum samples were the first blood sample of blood drawn at admission). The results showed that the serum calprotectin levels of patients with secondary pneumonia complications were significantly higher than that without pneumonia (median 5197.22 (IQR 3315.09-8560.17) versus median 3516.94 (IQR 2623.534509.64) pg/ml, P = .019; Fig 2B). And this result suggests that patients with high serum levels are more prone to pneumonia complications. According to univariate logistic regression analysis, as shown in the Table 3, all 3 factors of GCS score, Fisher
Figure 1. Correlation between serum calprotectin levels and clinical severity.
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Figure 2. Level of serum calprotectin concentrations in aSAH patients.
grade and serum calprotectin are related to the occurrence of pneumonia. For serum calprotectin, when serum level of calprotectin increased by 1 ng/ml, the unadjusted risk of pneumonia increased by 33.3% (OR, 1.333; 95% CI, 1.038-1.712; P < .001). But by the multivariate analysis, serum calprotectin cannot be used as an independent predictor of pneumonia (Table 2).
Relevance between Serum Calprotectin Level and Occurrence of DCI in aSAH Patients DCI is a common and serious complication after aSAH, which is related to the increase of disability and mortality. According to the DCI criteria, 19 of 54 patients developed DCI and 39 did not develop DCI. The serum calprotectin levels of these 19 people were compared with those of 39
Table 3. Univariate and multivariate logistic regression analysis of pneumonia after aneurysmal subarachnoid hemorrhage Univariate analysis
Age Gender GCS Hunt-Hess Fisher CRP, mg/L Calprotectin, ng/mL D-dimer, mg/L FEU Hematocrit, % Platelets, 109/L Hemoglobin, g/L Albumin, g/L Bilirubin, mmol/L Sodium, mmol/L Potassium, mmol/L Glucose, mmol/L Fibrinogen, g/L PT, s
Multivariate analysis
OR (95% CI)
P value
0.983 (0.929-1.041) 0.970 (0.313-3.003) 0.774 (0.629-0.963) 1.745 (0.958-3.179) 3.510 (1.579-7.801) 1.020 (0.993-1.048) 1.333 (1.038-1.712) 1.027 (0.892-1.182) 0.957 (0.884-1.037) 1.005 (0.995-1.015) 0.993 (0.966-1.022) 0.963 (0.850-1.091) 1.005 (0.884-1.142) 1.161 (0.960-1.403) 0.985 (0.257-3.778) 1.223 (0.921-1.623) 0.783 (0.393-1.561) 0.735 (0.334-1.620)
.559 .970 .016 .069 .002 .148 .024 .710 .283 .355 .640 .556 .941 .124 .982 .164 .488 .446
OR (95% CI)
P value
0.893 (0.684-1.165)
.404
2.194 (0810-5.946)
.122
1.211 (0.915-2.647)
.180
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Table 4. Univariate and multivariate logistic regression analysis of DCI after aneurysmal subarachnoid hemorrhage Univariate analysis
Age Gender GCS Hunt-Hess Fisher CRP, mg/L Calprotectin, ng/mL D-dimer, mg/L FEU Hematocrit, % Platelets, 109/L Hemoglobin, g/L Albumin, g/L Bilirubin, mmol/L Sodium, mmol/L Potassium, mmol/L Glucose, mmol/L Fibrinogen, g/L PT, s
Multivariate analysis
OR (95% CI)
P value
1.003 (0.948-1.061) 0.489 (0.151-1.579) 0.706 (0.575-0.865) 2.353 (1.262-4.390) 3.339 (1.517-7.350) 1.027 (1.003-1.053) 1.517 (1.181-1.948) 1.049 (0.922-1.194) 0.965 (0.880-1.057) 0.997 (0.987-1.006) 1.005 (0.977-1.033) 1.138 (0.983-1.318) 0.960 (0.880-1.047) 0.940 (0.783-1.129) 0.597 (0.157-2.266) 1.143 (0.889-1.471) 0.761 (0.374-1546) 0.576 (0.241-1.375)
0.920 0.231 0.001 0.007 0.003 0.030 0.001 0.467 0.441 0.486 0.731 0.082 0.353 0.509 0.448 0.297 0.450 0.214
OR (95% CI)
P value
0.662 (0.465-0.942) 0.797 (0.300-2.114) 1.154 (0.392-3.392) 1.013 (0.979-1.048) 1.584 (1.112-2.257)
.022* .648 .665 .455 .011*
*P < 0.05.
people, and the results showed that the level of serum calprotectin in patients with DCI was significantly higher than that in patients without DCI (median 6380.89 (IQR 4323.93-11358.94) versus median 3569.67 (IQR 2623.534926.02) pg/ml, P = .001; Fig 2C). In addition, as shown in Table 4, we also conducted a univariate analysis to investigate the independent predictive markers of DCI among the factors. The univariate analysis results showed that among the listed indicators, only GCS score, Fisher grade, Hunt-Hess grade, CRP, and calprotectin were significantly associated with poor prognosis. The analysis shows these variables are not collinear, so we further put them into in the multivariate logistic regression model. The results showed that, like GCS score (OR, 0.662; 95%CI, 0.465-0.942; P = .022), calprotectin (OR, 1.584; 95% CI, 1.112-2.257; P = .011) could be used as an independent predictor of DCI after aSAH. This meant that every 1 ng/ml increase in serum calprotectin as a continuous variable, the risk of adjusted poor outcomes increased by 58.4% and the unadjusted risk increased by 51.7%.
Sensitivity and Specificity of Calprotectin in Predicting DCI after aSAH CRP has been proved to be an effective predictor of SAH25-27 and the early phase CRP levels following SAH is related to delayed ischemia or angiographic vasospasm.28 We compare the sensitivity and specificity of calprotectin with CRP to show the ability of calprotectin to predict DCI after aSAH. The AUC value of calprotectin was 0.782 (95% CI, 0.649-0.883; P < .001; sensitivity: 68.42%,
specificity: 82.86%), while the AUC value of CRP was 0.696 (95% CI, 0.556-0.814; P = .011; sensitivity: 78.95%, specificity: 60.00%; Fig 3A, Table 5). ROC curve suggested that the serum calprotectin level on admission could predict the subsequent DCI, and its predictive value was similar to that of CRP (P = .387). The cutoff value of calprotectin for predicting DCI in patients with aSAH was 5275 pg/ml. Additionally, we also calculated the AUC values of Fisher grade, GCS score and Hunt-Hess scale to predict DCI, respectively (Fig 3B-D). As shown in Table 5, The AUC of GCS, Fisher grade, and Hunt-Hess scale were 0.831 (95% CI, 0.703-0.920; P < .001; sensitivity: 89.47%, specificity: 70.59%), 0.759 (95% CI, 0.624-0.865; P < .001; sensitivity: 78.95%, specificity: 63.86%) and 0.723 (95% CI, 0.585-0.836; P = .012; sensitivity: 68.42%, specificity: 74.29%), respectively. There were no significant differences between the AUC value of serum calprotectin and the AUC value of GCS score, Fisher grade, and Hunt-Hess score (P = .589, P = .821, P = .502, respectively). In the combined logistic regression model, calprotectin did not significantly improve the AUC of these neurological scores (P > .05).
Relevance between Serum Calprotectin Level and Prognosis of aSAH Patients According to the follow-up results of 3 months after discharge, 54 patients were further divided into good prognosis group and poor prognosis group. In our study, 28 patients had a poor prognosis (mRS 3-6) and 26 patients with good prognosis (mRS 0-2). Then the serum calprotectin levels were compared retrospectively between the 2
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Figure 3. Values of variables in predicting delayed cerebral ischemia in patients with aSAH.
groups (Serum samples were the first blood drawn at admission). The data showed that serum calprotectin levels were significantly higher in the poor prognosis group than that in the good prognosis group (median 6364.30 (IQR 4039.90-8930.76) versus median 2920.72 (IQR 2436.664613.73) pg/ml, P < .001), which indicated there were a significant difference between the 2 groups (Fig 2D). To investigate the main factors affecting the prognosis of patients with aSAH, we performed univariate analysis for the indexes listed in Table 6. The univariate analysis results showed that among the listed indicators, only GCS score, Fisher grade, Hunt-Hess grade, CRP, and calprotectin were significantly associated with poor prognosis. In addition, we confirm that these variables are not
collinearity, so they are included in the multivariate logistic regression model. The results showed that only calprotectin (OR, 1.775; 95% CI, 1.190-2.647; P = .005) emerged as an independent predictor of poor prognosis at 3 months. This meant that every 1 ng/ml increase in serum calprotectin as a continuous variable, the risk of adjusted poor outcomes increased by 77.5% and the unadjusted risk increased by 75.2%.
Sensitivity and Specificity of Calprotectin in Predicting 3-Month Prognosis Previous studies have shown that CRP could be used as an effective index to predict the prognosis of SAH.25
Table 5. Receiver operating characteristic curve analysis of factors predicting DCI among aSAH patients
Criterion Area under curve 95% confidence interval Sensitivity 95% confidence interval Specificity 95% confidence interval + likelihood ratio 95% confidence interval likelihood ratio 95% confidence interval P value
Calprotectin
CRP
GCS
Fisher
Hunt-Hess
>5274.66 0.782 0.649-0.883 68.42 43.3-87.4 82.86 66.4-93.4 3.99 1.8-8.8 0.38 0.2-0.8 Reference
>16.2 0.696 0.556-0.814 78.95 54.4-93.9 60.00 42.1-76.1 1.97 1.2-3.2 0.35 0.1-0.9 .387
12 0.831 0.703-0.920 89.47 66.9-98.7 70.59 52.5-84.9 3.04 1.8-5.2 0.15 0.04-0.6 .589
>2 0.759 0.624-0.865 78.95 54.4-93.9 62.86 44.9-78.5 2.13 1.3-3.5 0.33 0.1-0.8 .821
>2 0.723 0.585-0.836 68.42 43.4-87.4 74.29 56.7-87.5 2.66 1.4-5.0 0.43 0.2-0.8 .502
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Table 6. Univariate and multivariate logistic regression analysis of 3-month prognosis after aneurysmal subarachnoid hemorrhage Univariate analysis
Age Female GCS Hunt-Hess Fisher CRP, mg/L Calprotectin, ng/mL D-dimer Hematocrit, % Platelets, 109/L Hemoglobin, g/L Albumin, g/L Bilirubin, mmol/L Sodium, mmol/L Potassium, mmol/L Glucose, mmol/L Fibrinogen, g/L PT
Multivariate analysis
OR (95% CI)
P value
1.019 (0.966-1.076) 0.978 (0.332-2.878) 0.717 (0.584-0.880) 3.211 (1.586-6.500) 3.402 (1.589-7.281) 1.038 (1.007-1.070) 1.752 (1.253-2.541) 1.207 (0.980-1.486) 0.913 (0.807-1.034) 0.997 (0.987-1.006) 0.976 (0.945-1.009) 0.963 (0.850-1.091) 1.024 (0.960-1.091) 1.094 (0.916-1.308) 0.577 (0.151-2.196) 1.038 (0.818-1.319) 1.469 (0.744-2.902) 1.171 (0.547-2.506)
.490 .967 .001 .001 .002 .017 .001 .076 .152 .478 .148 .556 .475 .322 .420 .758 .268 .685
OR (95% CI)
P value
0.826 (0.567-1.203) 1.607 (0.538-4.802) 1.547 (0.452-5.291) 1.011 (0.974-1.050) 1.775 (1.190-2.647)
.320 .396 .487 .558 .005*
*P < 0.05.
Therefore, in this study, we used the ROC curve to measure the ability of serum calprotectin and CRP in predicting the prognosis of patients of aSAH patients with CRP. The AUC values of calprotectin and CRP were 0.809 (95% CI, 0.6790.903; P < .001; sensitivity: 53.57%, specificity: 96.15%), and 0.693 (95% CI, 0.553-0.811; P = .015; sensitivity: 74.29%, specificity: 76.92%), respectively (Fig 4A, Table 7). The ROC
curve showed that serum calprotectin level on admission statistically significantly predicted 3-month poor outcome of patients, and the predictive value of the calprotectin concentration was similar to that of CRP (P = .218). The ROC curve showed serum calprotectin concentrations greater than 6020 pg/ml predicted poor prognosis at 3 months after discharge. In other words, the cutoff value of calprotectin in
Figure 4. Values of variables in predicting poor outcome in patients with aSAH.
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Table 7. Receiver operating characteristic curve analysis of factors predicting 3-month poor outcomes among aSAH patients
Criterion Area under curve 95% confidence interval Sensitivity 95% confidence interval Specificity 95% confidence interval + likelihood ratio 95% confidence interval likelihood ratio 95% confidence interval P value
Calprotectin
CRP
GCS
Fisher
Hunt-Hess
>6.02 0.809 0.679-0.903 53.57 33.9-72.5 96.15 80.4-99.9 13.93 2.0-98.2 0.48 0.3-0.7 Reference
>21.7 0.693 0.553-0.811 64.29 44.1-81.4 76.92 56.4-91.0 2.79 1.3-5.9 0.46 0.3-0.8 .218
22 0.782 0.647-0.884 71.43 51.3-86.8 72.00 50.6-87.9 2.55 1.3-5.0 0.40 0.2-0.7 .747
>2 0.766 0.631-0.870 75.00 55.1-89.3 73.08 52.2-88.4 2.79 1.4-5.4 0.34 0.2-0.7 .543
>2 0.769 0.635-0.873 60.71 40.6-78.5 80.77 60.6-93.4 3.16 1.4-7.3 0.49 0.3-0.8 .626
predicting poor prognosis in patients with aSAH was 6020 pg/ml. In addition, neurological scores are often used to predict prognosis. Here, we calculated the AUC values of Fisher grade, GCS score, and Hunt-Hess scale, respectively in the 54 patients (Fig 4BCD). As shown in Table 5, The AUC of Fisher grade, GCS, and Hunt-Hess scale were 0.766 (95% CI, 0.631-0.870; P = .008; sensitivity:75.00%, specificity:73.08%), 0.782 (95% CI, 0.647-0.984; P = .004; sensitivity:71.43%, specificity:72.00%), and 0.769 (95% CI, 0.635-0.873; P = .007; sensitivity:60.71%, specificity:80.77%). In terms of prognostic predictive ability assessed by AUC, there were no substantial differences between serum calprotectin concentrations and other variables, including GCS score (P = .747), Fisher grade (P = .543), and Hunt-Hess scale (P = .626). In the combined logistic regression model, calprotectin statistically significantly improved the AUC of GCS score (P = .031) and Hunt-Hess scale (P = .021) but did not statistically significantly improve the AUC of Fisher grade (P = .073).
Discussion The correlation between serum concentrations of calprotectin (S100A8/A9) and inflammation has been well described. The S100A8/A9 complex has been proved to be a diagnostic marker of inflammation, especially in noninfectious inflammatory diseases such as arthritis, chronic inflammatory lung disease, and intestinal disease.29 Calprotectin mainly exists in the cytoplasm of inflammatory cells such as granulocytes and monocytes. In the central nervous system, calprotectin is abundantly expressed in microvascular endothelial cells and microglial cells.30 However, there is no report on the relevance between calprotectin and aSAH. We firstly reported that serum calprotectin levels were significantly elevated in aSAH patients within 48 hours after onset compared with the healthy controls. According to our statistical results, calprotectin levels increased significantly within 12 hours after onset, peaked at approximately 24-36 hours, and then decreased again between 36 and 48 hours. This
phenomenon indicates that calprotectin is a marker of acute inflammatory response in SAH patients. Further retrospective statistical analysis showed that patients with subsequent pneumonia complications had significantly higher serum calprotectin levels on admission than patients without pneumonia complications, but in the multivariate logistics analysis, serum calprotectin cannot be used as an independent predictor of pneumonia. However, the statistical results showed that, like GCS score and Fisher grade, serum calprotectin was related to the occurrence of pneumonia, which suggests that calprotectin is an early marker of inflammation in SAH patients and it is associated with the complications of secondary pneumonia. This may be related to the role of calcitonin in the pathogenesis of pneumonia. It was reported that high level of S100A8 and S100A9 in the alveolar walls of lungs of mice infected with S. pneumoniae involved in the transepithelial migration of macrophages and neutrophils31 and calprotectin has a strong link between local alveolar inflammation, neutrophil activation, and NETosis.32 Moreover, it has been reported that calprotectin is an endogenous ligand of toll-like receptor 4 (TLR4) and of the receptor for advanced glycation end products (RAGE)33 .Calprotectin also activates the MAPK and NFkB signaling pathways, which leads to the amplification of the proinflammatory cascade through TLR4 and RAGE pathways.34 Coincidentally, in the early stage of SAH, the main activation of signaling pathway is TLR4-related pathway,35 and RAGE is also directly involved in the inflammatory response after SAH.36 Based on these studies, we speculate that as an endogenous ligand for TLR4 and RAGE, calprotectin significantly increased in the early stage of SAH and bind to TLR4 and RAGE, thus participating in the activation of TLR4 and RAGE signal transduction pathways. Recent studies have shown that DCI after aSAH is associated with vasospasm, microcirculation disorder and microthrombosis, cortical diffusion inhibition, and inflammation. In our study, analyzed by multivariate logistic regression, the result showed that calprotectin can be used as an
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independent predictor of DCI emergence. Moreover, the results of ROC curve showed that the predictive ability of calprotectin to DCI was similar to that of other standard clinical scoring systems such as Fisher grade, GCS score, Hunt-Hess score, and similar to that of CRP. The exact mechanism by which calprotectin is involved in DCI is unclear, but it has been reported that the calprotectin can bind to arachidonic acid (AA), the calprotectin-AA complex promotes the transcellular transport of AA, which leads to vascular endothelial injury and vascular inflammation.37,38 In addition, the transfer of AA can promote the activation of NADPH oxidase to produce ROS in phagocytes.39 Our statistical results showed that, by multivariate logistic regression model, among the factors associated with 3-month poor prognosis of aSAH, only the calprotectin is an independent predictor of poor prognosis at 3 months (P = .005). There may be many mechanisms by which calprotectin affects prognosis including consciousness level and limb motor function in patients with SAH, but we think there are 2 most important points, 1 of which is cerebral vasospasm and another mechanism is neuronal cell death. The onset of aSAH elicits activation of the inflammatory cascade, and ongoing neuroinflammation leads to secondary complications, such as vasospasm, DCI, and neuronal cell death. And these secondary complications have been considered to be the causes of neurocognitive impairment.1 Calprotectin is involved in the inflammatory cascade and has a wide range of proinflammation effects, therefore high serum calprotectin correlates with poor outcome. Furthermore, our data analysis showed that the calprotectin has a good accuracy in predicting adverse outcome. The AUC of the calprotectin concentration was similar to those of CRP, Fisher grade, GCS score, and Hunt-Hess scale for the prediction of these poor outcomes, while in the combined logistic-regression models, calprotectin improved the predict ability of clinical scores. These further analyses all revealed that serum level of calprotectin was independently associated with poor outcome and suggested that calprotectin can be a good predictor associated with severity and prognosis of aSAH. In fact, calprotectin has been studied as a biomarker in several diseases. Calprotectin has been found to be associated with the progression of inflammatory bowel disease. And fecal calprotectin can predict and monitor the risk of recurrence in patients with inflammatory bowel disease after treatment.40 Besides, calprotectin has been found to play an important role in the rheumatoid arthritis, and plasma calprotectin has the strongest correlation with assessments of disease activity.11 In patients undergoing cardiac surgery, plasma calprotectin was significantly increased, and can predict the onset of acute kidney injury.41 S100A9 knockout in mice reduces TBI-induced neuroinflammation, protect the brain from secondary damage by TBI, and improves functional outcome after TBI.42
We propose for the first time that the level of serum calprotectin can be used as an independent predictor of DCI after aSAH and the prognosis of aSAH and serum calprotectin is not only an objective indicator, but also a new and effective target for the treatment of aSAH. Anticalprotectin antibodies and their antagonists may be used in the treatment of SAH. However, our current study still has some limitations. First, we did not continue to track the changes in calprotectin over a longer period. We are limited to talking about acute inflammatory cascade of aSAH. However, if peripheral blood and cerebrospinal fluid can be obtained from the same patients at different time points, serial change of calprotectin concentrations can be studied. Second, we are limited to talking about 3month prognosis. If there were longer follow-up records, we would be able to discuss the relationship between aSAH mortality rate and calprotectin. And our conclusions still need to be validated in a larger population of multicenter. Last but not least, the cause of elevated serum in patients with aSAH and its mechanism in the pathology of SAH should be further in-depth studied.
Conclusion In conclusion, serum calprotectin level within 48 hours after onset in patients with aSAH has high predictive value of prognosis and complications. Serum calprotectin could be used as an independent predictor of poor prognosis at 3 months as well as DCI emergence. Serum calprotectin level was related to the occurrence of secondary pneumonia complications after aSAH but cannot be used an independent predictor of secondary pneumonia. Therefore, it may become a prognostic biomarker of aSAH. Declaration of Interest: financial interests.
The authors declare no competing
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