Pentraxin 3 as a Prognostic Biomarker in Aneurysmal Subarachnoid Hemorrhage

Introduction

Aneurysmal subarachnoid hemorrhage (aSAH) remains one of the most critical and life-threatening cerebrovascular disorders, contributing significantly to neurological morbidity and mortality worldwide [1,2]. Despite advancements in diagnostics and therapeutic interventions, the prognosis of patients with aSAH remains poor [3]. About 10% of patients die before reaching medical attention, while another 45% die within 1 month after the hemorrhagic event [4,5]. These statistics underscore the urgency and clinical importance of improving our understanding of this complex disorder [3,6]. Among the multiple complications arising from aSAH, cerebral vasospasm and delayed cerebral ischemia (DCI) are particularly detrimental. DCI, characterized by reduced cerebral blood flow, often leads to neuronal injury and is a major cause of long-term neurological deficits and poor outcomes in aSAH patients [7]. These pathological events commonly appear on the third day after hemorrhage, peak around the seventh day, and can persist for as long as 2–3 weeks [8]. Such manifestations compound the patient’s clinical condition, often resulting in dire neurological deficits or even death [9,10]. The severity of aSAH is commonly assessed using clinical grading systems, such as the Hunt-Hess classification, the World Federation of Neurosurgical Societies (WFNS) scale, and the modified Fisher (mFisher) grading system [11]. These scoring systems are instrumental in determining the initial severity, predicting clinical outcomes, and guiding therapeutic interventions. However, despite their utility, reliable biomarkers that can complement or enhance the predictive power of these scales are still in need of exploration [12].

In the context of advancing medical science, the role of neuroinflammation in the progression and pathophysiology of aSAH has gained growing attention. Neuroinflammation is critical in neuronal damage and clinical outcomes, making it central to emerging research [13]. Researchers have become increasingly interested in identifying inflammatory markers that can serve as reliable indicators for predicting disease severity and prognosis in aSAH [14,15]. Among the array of inflammatory markers, pentraxin 3 (PTX3) stands out due to its unique role in both innate immunity and the modulation of inflammatory cascades, particularly in cerebrovascular and cardiovascular conditions [16,17]. PTX3 is a long pentraxin that is an essential element in innate immunity. It can bind to various microbial moieties and assist in opsonization, making it a sentinel in the body’s first line of defense [18]. Beyond its role in innate immunity, PTX3 has also been demonstrated to be a modulator of inflammatory cascades in cardiovascular and cerebrovascular conditions. In these vascular diseases, elevated PTX3 levels have been correlated with disease severity and have been investigated as a prognostic marker [19]. Despite these compelling developments, the application of PTX3 as a biomarker in aSAH has not been extensively studied, particularly regarding its levels in cerebrospinal fluid (CSF) [20]. This presents an evident gap in our understanding of aSAH, especially given PTX3’s established role in other vascular and inflammatory conditions [21]. Several questions remain unanswered, including whether CSF PTX3 levels could be used as a reliable gauge of disease severity or if they are indicative of long-term clinical outcomes, including mortality and neurological deficits [22].

However, the relationship between PTX3 levels in CSF and its role in predicting the clinical severity and long-term prognosis in aSAH patients remains relatively unexplored. The primary objective of this study was to assess the correlations between PTX3 concentrations in the CSF and various clinical and prognostic parameters in patients with aSAH, especially those undergoing endovascular coiling within 24 hours after the hemorrhage.

Material and Methods

STUDY DESIGN:

This retrospective study, conducted from March 2020 to March 2023 at our hospital, focused on 65 patients who underwent endovascular coiling for aSAH. Patients were included if they were admitted within 24 hours of symptom onset, diagnosed with aSAH, received surgical intervention within 24 hours after admission, and were 18–70 years of age. Exclusion criteria were a history of neurological diseases, recent surgeries, acute or chronic infectious diseases, use of certain medications, and presence of other systemic diseases. A control group of 20 patients, treated for other diseases and undergoing spinal anesthesia, was also included. CSF was collected for PTX3 level assessment. Informed consent was obtained from all subjects. Our study received ethics clearance from our hospital’s ethics committee (IS230519, 2023-09-27) and was conducted in strict adherence to the Declaration of Helsinki and applicable guidelines. We maintained the highest ethical standards in the design, execution, and reporting phases. Data confidentiality was rigorously upheld, and personal identifiers were anonymized to ensure privacy protection.

DIAGNOSTIC CRITERIA FOR ANEURYSMAL SUBARACHNOID HEMORRHAGE:

In the study group, the diagnosis of aSAH was established in accordance with clinical guidelines [23]. The initial diagnostic step involved performing a non-contrast head CT scan, which is widely recognized as the first-line imaging modality due to its high sensitivity in detecting acute hemorrhage. In cases where the CT scan results were inconclusive and clinical suspicion remained high, a lumbar puncture was performed at least 12 hours after the onset of symptoms to assess for xanthochromia in the cerebrospinal fluid, which supports the diagnosis of subarachnoid hemorrhage. Following the confirmation of hemorrhage, further vascular imaging was performed to identify the presence, location, and morphology of an aneurysm. Computed tomography angiography (CTA) was used as a non-invasive method for the initial evaluation of cerebral vasculature. In selected cases where detailed vascular anatomy was required or when CTA findings were ambiguous, digital subtraction angiography (DSA) was performed, given its status as the criterion standard for aneurysm detection and characterization.

CLINICAL EVALUATION AND PATIENT MANAGEMENT:

All data were retrospectively extracted from the hospital’s electronic medical records database. Upon admission, the severity of each patient’s condition was evaluated using established clinical grading systems. The Hunt-Hess classification [24] was employed, with grading defined as follows: Grade 0 for unruptured aneurysms; Grade 1 for patients who were asymptomatic or experienced only a mild headache and slight neck stiffness; Grade 2 for those with moderate to severe headache and neck stiffness, without neurological deficits aside from possible cranial nerve palsy; Grade 3 for patients exhibiting somnolence, confusion, or minor focal neurological deficits; Grade 4 for those in a state of stupor with moderate to severe hemiparesis, and potential early signs of brain herniation accompanied by autonomic dysfunction; and Grade 5 for patients in deep coma, with evidence of brain herniation and near-death conditions.

We used the World Federation of Neurosurgical Societies (WFNS) grading system [25] to further assess patient status based on the Glasgow Coma Scale (GCS) and the presence of motor deficits. Specifically, WFNS Grade 1 was assigned to patients with a GCS score of 15 and no motor deficits; Grade 2 to those with a GCS score of 13–14 without motor deficits; Grade 3 to patients with a GCS score of 13–14 accompanied by motor deficits; Grade 4 to those with a GCS score of 7–12, regardless of motor function; and Grade 5 to patients with a GCS score of 3–6, with or without motor deficits.

To quantify the volume of subarachnoid hemorrhage as visualized on computed tomography (CT) scans, the modified Fisher (mFisher) grading scale [26] was applied. The mFisher grading is defined as follows: Grade 0 indicates the absence of subarachnoid or intraventricular hemorrhage; Grade 1 represents a small or thin layer of subarachnoid hemorrhage without bilateral intraventricular hemorrhage; Grade 2 denotes a small or thin layer of subarachnoid hemorrhage accompanied by bilateral intraventricular hemorrhage; Grade 3 is indicative of extensive subarachnoid hemorrhage without bilateral intraventricular hemorrhage; and Grade 4 corresponds to extensive subarachnoid hemorrhage with bilateral intraventricular hemorrhage.

Additional clinical data, including sex, age, average arterial pressure, and blood sugar levels at admission, were recorded. Information regarding the aneurysm’s size, location, and treatment-related complications – such as acute hydrocephalus and pulmonary infection – were also collected.

PATIENT FOLLOW-UP AND OUTCOME GROUPING:

All patients were followed up for 3 months after discharge, and their clinical outcomes were evaluated using the Modified Rankin Scale (mRS). The mRS scoring system was defined as follows:

For the purpose of analysis, an mRS score of less than 3 was considered indicative of a good prognosis, whereas an mRS score of 3 or greater was considered indicative of a poor prognosis.

PTX3 LEVEL MEASUREMENT IN CSF:

CSF samples (5 ml) were collected from all aSAH patients within 24 hours after surgery and from the control group during spinal anesthesia. After allowing the samples to rest for 20 minutes, they were centrifuged at 3000 rpm for 15 minutes, and the resulting supernatant was stored at −80°C until further analysis. PTX3 levels were quantified using an enzyme-linked immunosorbent assay (ELISA) kit from Zhengzhou Cybelle Bio-Technology Co., Ltd, in strict accordance with the manufacturer’s protocol. The assay utilized a sandwich ELISA technique, in which a specific monoclonal antibody against human PTX3 was employed as the capture antibody, and a biotinylated anti-PTX3 antibody was used as the detection antibody. Positive controls, comprising known concentrations of recombinant human PTX3, and negative controls (assay buffer without sample) were included in each assay to ensure the reliability of the method. Optical density was measured at 450 nm using a calibrated microplate reader. A standard curve was generated from serial dilutions of the PTX3 standards provided with the kit, and the concentrations of PTX3 in the CSF samples were determined by interpolation from this curve. All samples were measured in duplicate, and the intra- and inter-assay coefficients of variation were maintained below 10%, ensuring the reproducibility and precision of the assay results.

STATISTICAL ANALYSIS:

Data were processed using SPSS version 27.0 and MedCalc version 19.7.2 software packages. For categorical variables, frequencies and percentages were calculated and analyzed using the chi-square test (χ²). For continuous variables that were normally distributed, the mean and standard deviation were reported and subjected to t tests. For continuous data that did not follow a normal distribution, median values and interquartile ranges were used, and the Mann-Whitney U test was employed for statistical analysis. Bivariate correlation analysis was performed using Spearman’s rank correlation coefficient. Receiver operating characteristic (ROC) curve analysis was conducted to evaluate the diagnostic value of cerebrospinal fluid PTX3 levels and various scales for poor prognosis in aSAH patients. Multiple logistic regression was utilized to assess independent risk factors for unfavorable clinical outcomes in aSAH patients. Variables that were statistically significant in univariate analyses were included in the multivariate model, and odds ratios (ORs) along with 95% confidence intervals (95% CIs) were calculated. A P value of less than 0.05 was considered statistically significant.

Results

CHARACTERISTICS OF THE STUDY POPULATION:

We evaluated clinical characteristics associated with prognosis in 65 patients with aneurysmal SAH, categorizing them into poor (n=31) and good (n=34) prognosis groups. Four patients (6.2%) in the overall cohort died during the 3-month follow-up. The causes of death were consistent with common complications of aneurysmal SAH and included: 1 case of rebleeding, 1 case of severe cerebral vasospasm leading to secondary infarction, 1 case of pneumonia-related respiratory failure, and 1 case of brain herniation resulting from elevated intracranial pressure. Key findings indicate significant differences in several clinical parameters. Patients with poor prognosis had higher mean arterial pressure (MAP) (114 mmHg vs 110 mmHg; P=0.032) and a higher incidence of acute hydrocephalus (32.3% vs 11.8%; P=0.045). Additionally, pneumonia was more prevalent in the poor prognosis group (58.1% vs 23.5%; P=0.005), suggesting it can contribute to worse outcomes. Furthermore, levels of PTX3 were significantly elevated in patients with poor prognosis (2.340±0.980 ng/ml vs 1.460±0.564 ng/ml; P<0.001). These findings highlight the importance of monitoring MAP, the presence of hydrocephalus, pneumonia, and PTX3 levels to inform prognosis and guide management in SAH patients (Table 1).

ASSOCIATIONS BETWEEN PTX3 LEVELS AND CLINICAL SEVERITY GRADING SYSTEMS:

Subjects in the control group had no detectable levels of PTX3 in the CSF. Conversely, patients diagnosed with aSAH had significantly higher PTX3 levels. Quantitatively, the data demonstrated zero ng/ml of PTX3 in the control group versus 1.916±1.058 ng/ml in the aSAH group, a finding that was statistically significant (P<0.001). Moreover, our results showed a positive correlation between PTX3 levels in the CSF and the clinical grading scales used upon admission, namely the Hunt-Hess, WFNS, and mFisher grading systems. As these grading scores increased, indicating a more severe aSAH condition, the levels of PTX3 in the CSF also rose proportionally (Table 1). This observation could suggest that PTX3 levels could serve as a sensitive indicator for evaluating the severity of aSAH in a clinical setting. The observed elevation in PTX3 levels in aSAH patients indicates that this pentraxin may serve as a potential biomarker for early diagnosis or prognosis.

RISK FACTORS ASSOCIATED WITH POOR PROGNOSIS AT 3-MONTH FOLLOW-UP IN ASAH PATIENTS:

Our 3-month follow-up identified 31 patients with poor prognostic outcomes. Compared to the good prognosis group, these patients had significantly higher levels of PTX3 in their CSF (P<0.001). Furthermore, elevated Hunt-Hess, WFNS, and mFisher scores, as well as higher mean arterial pressure, were observed at the time of admission (P<0.05). Higher rates of acute hydrocephalus and pulmonary infection were also noted during the treatment phase in this group (P<0.05). The ROC curve analysis revealed a large area under the curve for PTX3 levels in CSF in predicting poor prognosis, with an optimal diagnostic cut-off value of 1.990 ng/ml. Interestingly, the prognostic utility of CSF PTX3 levels was not significantly different from Hunt-Hess, WFNS, and mFisher scores (P>0.05). There were no significant differences between the 2 groups in terms of age, sex, admission blood glucose levels, and aneurysm location and size (P>0.05; Tables 2, 3).

UNIVARIATE AND MULTIVARIATE REGRESSION ANALYSES OF STATISTICALLY SIGNIFICANT PARAMETERS:

To identify independent risk factors for poor prognosis at the 3-month follow-up in patients with aSAH, univariate and multivariate regression analyses were conducted. The variables analyzed included Hunt-Hess grade, WFNS grade, mFisher grade, mean arterial pressure, CSF PTX3 levels, acute hydrocephalus, and pulmonary infection, all of which had shown statistical significance in preliminary analyses. Our analyses revealed that a CSF PTX3 level of 1.990 ng/ml or higher, a WFNS grade of 3 or higher, and a Hunt-Hess grade of 3 or higher were independent risk factors for a poor prognosis at 3 months. These findings suggest that monitoring these parameters can be invaluable in predicting patient outcomes. The robustness of the independent variables, such as PTX3, WFNS, and Hunt-Hess grades, underscores their utility as reliable predictors (Tables 4, 5).

POST HOC POWER ANALYSIS AND SAMPLE SIZE ADEQUACY:

A post hoc power analysis was performed to assess the adequacy of the sample size for detecting significant differences in cerebrospinal fluid (CSF) PTX3 levels between patients with poor and good prognosis. Based on the observed mean PTX3 levels (2.340±0.980 ng/ml in the poor prognosis group vs 1.460±0.564 ng/ml in the good prognosis group), the effect size (Cohen’s d) was calculated as 1.12, indicating a large difference between groups. With an alpha of 0.05 and 80% statistical power, the required sample size for detecting significant differences was 18 participants per group. The study included 31 patients in the poor prognosis group and 34 in the good prognosis group, exceeding the minimum required sample size. Thus, the study was sufficiently powered to detect statistically significant differences in PTX3 levels, supporting the robustness of the findings and ensuring that observed differences were unlikely to be due to random chance.

Discussion

Recent studies, together with our findings, provide a more comprehensive insight. Taylor et al [27] identified inflammasome proteins (caspase-1, ASC, IL-18, IL-1β) as promising inflammatory biomarkers for aSAH prognosis in serum and CSF, demonstrating significant correlations with worse outcomes. Similarly, our study shows that elevated CSF PTX3 levels correlate with initial disease severity and independently predict poor 3 month outcomes. However, while inflammasome proteins reflect upstream inflammatory activation, PTX3 may be a more specific indicator of sustained inflammatory burden and endothelial dysfunction. Moreover, our focus on CSF-derived biomarkers provides a direct assessment of central nervous system inflammation, potentially yielding greater prognostic accuracy. Ho et al [28] reviewed CSF metabolomic alterations in aSAH, highlighting biomarkers such as lactate, pyruvate, glucose, and glutamate as indicators of ischemic damage and poor outcomes, yet they did not establish a specific routine biomarker. In contrast, our study supports PTX3 as a well-defined inflammatory biomarker with clear prognostic value, validated by quantitative analysis and ROC metrics. PTX3 offers direct insight into the inflammatory response, making it a practical early predictor of disease progression. Wu et al [29] reported that CSF sST2 levels peak within 2 days after aSAH and correlate with poor prognosis, particularly regarding cerebral edema (AUC=0.988). Similarly, our findings demonstrate that CSF PTX3 is an independent predictor of poor functional outcomes and correlates significantly with clinical severity scales (Hunt-Hess, WFNS, mFisher). Although sST2 reflects cerebral edema and systemic inflammation, PTX3 directly regulates vascular inflammation, a mechanism more intimately associated with aSAH severity. Together, these comparisons underscore PTX3’s potential to refine biomarker-based prognostic models in aSAH. Our findings demonstrate that PTX3 correlates with clinical severity and prognosis, supporting its use as a dynamic biomarker for disease progression. In clinical practice, daily PTX3 assessments during the acute phase, followed by weekly measurements in recovery, could provide real-time insights into inflammatory responses and guide timely interventions. Incorporating PTX3 testing into standard protocols, alongside existing grading systems like Hunt-Hess and WFNS, would enhance prognostic accuracy and enable tailored management strategies. While initial testing costs may be a consideration, PTX3 could optimize resource allocation by identifying high-risk patients for targeted interventions, reducing complications, and improving long-term outcomes. Ultimately, PTX3 testing could facilitate personalized treatment approaches, enhancing both clinical outcomes and cost-effectiveness.

PTX3, categorized as an acute-phase reactant, offers critical insights into the underlying pathophysiological processes mediated by the innate immune system. It is synthesized de novo by a myriad of cells integral to the immune response, such as macrophages, dendritic cells, and neutrophils, as well as by endothelial cells, fibroblasts, and adipocytes that participate in tissue remodeling and repair [30,31]. This wide range of cell types involved in PTX3 synthesis underscores its diverse roles in immune responses and vascular biology. Under basal physiological conditions, PTX3 is conspicuously absent or present in negligible amounts in the adult human brain [32]. However, its rapid upregulation during inflammatory conditions or tissue injury signifies a highly dynamic and responsive nature. Specifically, elevated PTX3 levels serve as a reliable marker for the severity and progression of inflammatory states, thus offering prognostic utility [33,34]. The acute-phase kinetics of PTX3 are particularly intriguing, given its swift and abundant release following an inflammatory stimulus, which underscores its potential as an early diagnostic biomarker. Moreover, PTX3 has been shown to interact with a range of ligands, including microbial components and apoptotic cells, thereby modulating the efficacy of phagocytic activity and innate immune responses. This in turn impacts vascular inflammatory responses that are pivotal in pathological conditions such as acute myocardial infarction, atherosclerosis, and large-vessel vasculitis [35,36]. In ischemic stroke models, PTX3 has been shown to facilitate post-injury vascular-neural repair. Elevated plasma PTX3 levels have been correlated with poor prognosis in acute cerebral infarction patients [37–40].

Our study demonstrated that the CSF levels of PTX3 increased in the early phase of aSAH, further elevating in patients who developed vasospasm. Importantly, elevated PTX3 levels were associated with greater bleeding volume and worse disease severity. The rapid lysis of red blood cells deposited in the subarachnoid space after aSAH could cause sterile inflammation by releasing hemoglobin, thereby stimulating glial cells, among others, to produce PTX3. The CSF PTX3 levels were particularly higher in patients with elevated Hunt-Hess, WFNS, and mFisher grades, indicating a direct correlation between PTX3 levels, the extent of subarachnoid hemorrhage, and the severity of the clinical condition. Moreover, patients with elevated PTX3 levels at admission had generally poorer clinical outcomes, suggesting that CSF PTX3 is an independent risk factor for unfavorable prognosis in aSAH patients. Clinically, these traditional grading systems can be subjected to inter-observer variability due to subjective assessments. The incorporation of PTX3 levels as an objective biomarker could therefore provide a more robust and reliable criterion for patient evaluation. Additionally, continuous monitoring of CSF PTX3 levels during the treatment course could facilitate dynamic assessment of the patient’s condition, thus enhancing the overall management strategy.

The threshold value of 1.990 ng/ml for PTX3 was determined through receiver operating characteristic (ROC) curve analysis, a robust method for identifying the optimal cut-off point for diagnostic and prognostic markers. This threshold maximized the Youden index (0.480), providing the best balance between sensitivity (70.0%) and specificity (78.0%) for predicting poor prognosis in a cohort of aSAH patients. While alternative thresholds were tested, none demonstrated superior diagnostic performance, underscoring the reliability of this value in distinguishing between favorable and poor outcomes. However, we acknowledge that the optimal PTX3 threshold may vary across different populations, and further studies in larger, multicenter cohorts are warranted to refine and validate this cut-off for broader clinical applicability. The focus on patients undergoing endovascular coiling within 24 hours of symptom onset in aSAH is critical, as early intervention significantly improves outcomes by reducing rebleeding risk and complications such as vasospasm and delayed cerebral ischemia. By minimizing the time to intervention, this approach stabilizes patients more quickly and reduces hemorrhage-related damage, enhancing long-term recovery prospects. This study’s design, centered on early treatment, eliminates confounding variables related to delayed interventions, providing a clearer evaluation of PTX3’s prognostic value in a more homogenous group, thus allowing for a more accurate assessment of its predictive potential. Regarding the absence of detectable PTX3 in the control group, consisting of patients undergoing spinal anesthesia, this finding highlights PTX3’s role as a marker of acute inflammatory responses. In contrast to aSAH patients, who experience significant neuroinflammation due to hemorrhagic injury and subsequent surgical intervention, the control group lacked similar inflammatory stimuli, explaining the absence of PTX3. This stark difference supports the specificity of PTX3 as a biomarker for aSAH, correlating with both inflammatory processes and clinical severity. Our findings are consistent with previous studies demonstrating that PTX3 levels are elevated in acute neurological injuries, further validating its potential as a useful biomarker for early detection and prognosis in aSAH.

Our study has several limitations. First, its observational design and lack of mechanistic experiments prevent establishing a direct causal link between elevated CSF PTX3 levels and aSAH severity or progression. Second, despite multivariate adjustments, residual confounders such as genetic variations, comorbidities, and unmeasured biomarkers may have influenced our findings, highlighting the need for propensity score matching and prospective validation. Third, the limited generalizability of our cohort and absence of longitudinal data constrain the evaluation of PTX3 as a marker for long-term outcomes, including functional recovery and cognitive impairment. Fourth, due to practical and ethical constraints, the control group comprised patients undergoing lumbar puncture as part of their clinical management rather than normal, healthy individuals. In clinical practice, normal individuals rarely undergo cerebrospinal fluid sampling. This approach may limit the representativeness of the control group, as these patients, with unspecified neurological disease, might not fully reflect a normal population.

Conclusions

We found that cerebrospinal fluid levels of PTX3 within 24 hours following endovascular embolization in patients with aSAH are closely correlated with initial Hunt-Hess, WFNS, and mFisher grades. Additionally, PTX3 levels are independently associated with poor 3-month outcomes. These findings establish PTX3 as a potential inflammatory biomarker for assessing the severity and prognosis of aSAH.