Assessment of Brain-Specific and Neuroendocrine Biomarkers for Intracerebral Hemorrhage Diagnosis: A Prospective Cohort Study

Abstract

Background and Purpose

The timing of diagnosis is a critical factor that significantly impacts medical treatment and patient outcomes in individuals with intracerebral hemorrhage (ICH). The study primarily aimed to evaluate the diagnostic potential of four brain-specific biomarkers, glial fibrillary acidic protein (GFAP), neurofilament light chain (NfL), tau, and ubiquitin C-terminal hydrolase-L1 (UCH-L1) to differentiate CT-confirmed ICH patients from healthy controls. Secondary aims included comparing results between younger (18-59 years) and older (≥60 years) ICH patients and exploring neuroendocrine hormone levels (adrenocorticotropic hormone [ACTH], agouti-related peptide [AgRP], ciliary neurotrophic factor [CNTF], and growth hormone [GH]) as potential diagnostic tools.

Methods

Serum samples were collected from consecutive adult patients with CT-confirmed ICH diagnoses on days 1 (first 24 hours), 2, 3, 7, and 14 for biomarker analyses in a pilot study. A total of 71 patients and 30 healthy controls were included in the analysis.

Results

Serum concentrations of four brain-specific biomarkers demonstrated significant differentiation between ICH patients and controls at all assessed time points (Days 1, 2, 3, 7, and 14; all p<0.05), with similar significant elevations observed for NfL, tau, and UCH-L1. Day 1 GFAP levels were markedly higher in ICH patients (median 9598 pg/mL compared with healthy controls (39.2 pg/mL; p<0.05). No significant differences were found between young and aged adult ICH subgroups. Neuroendocrine ACTH and AgRP levels were significantly higher in ICH patients compared to controls on both measured time points (Days 1 and 7).

Conclusions

The study’s findings suggest that GFAP, NfL, tau, UCH-L1, ACTH, and AgRP exhibit potential as biomarkers for differentiating spontaneous ICH patients from healthy controls. However, given the limitations of the current study design, these findings should be interpreted with caution and cannot be expected to translate directly into clinical practice. Further research is needed to assess these biomarkers’ roles in distinguishing stroke types and predicting ICH outcomes.

Introduction

Acute spontaneous intracerebral hemorrhage (ICH), a form of hemorrhagic stroke, is characterized by non-traumatic bleeding within the brain parenchyma and affects nearly 3 million individuals worldwide annually. ¹ ICH has a high mortality rate, with half of the deaths occurring within the first 48 hours, leaving most survivors with long-term disability and imposing a substantial economic burden. ² While the primary focus of ICH is on the damage caused by bleeding in the brain, it can indeed have secondary effects on other parts of the central nervous system (CNS), including the pituitary gland and hypothalamus, through factors such as ischemia, inflammation, excitotoxicity, or immunity. ³

Despite the critical importance of timing in diagnosis for influencing medical treatment and patient outcomes, the non-contrast cranial CT scan remains the gold standard for diagnosing acute ICH; however, limitations in availability and the need for rapid triage tools motivate interest in blood-based biomarkers. ⁴ The increasing use of cranial computed tomography (CT) in the emergency department for diagnosing suspected ICH has been accompanied by concerns about its costs, limited diagnostic value in non-trauma patients with symptoms mimicking ICH, and the significant constraint posed by its unavailability in certain settings. ⁵ Identifying a blood-based brain injury marker could facilitate the early diagnosis of ICH, differentiation from mimicking diseases, and monitoring treatment efficacy.

ICH causes glial and neuronal cell death, which could potentially lead to increased serum levels of brain injury markers such as glial fibrillary acidic protein (GFAP), neurofilament light chain (NfL), ubiquitin carboxy-terminal hydrolase-L1 (UCH-L1), spectrin, myelin basic protein, and tau in the systemic circulation.^6-9 Although only a few studies have evaluated the diagnostic value of NfL, tau, and UCH-L1,^10-13 GFAP remains one of the most extensively studied brain injury markers in ICH patients.^14-20 Notably, the combination of GFAP and UCH-L1 biomarkers led to their approval by the U.S. Food and Drug Administration for use in traumatic brain injury (TBI) patients in 2018.^6,11,21

The selection of GFAP, NfL, tau, and UCH-L1 was based on their established roles in acute central nervous system (CNS) injury, particularly within the traumatic brain injury (TBI) literature, where they have been extensively studied and validated as blood-based biomarkers of glial and neuronal injury.^6,22,23 Prior work from our group and others has demonstrated the temporal sensitivity and translational relevance of these markers in acute brain injury contexts.^6,22,23 Importantly, GFAP and UCH-L1 have achieved regulatory clearance for clinical use in TBI, underscoring their robustness as circulating biomarkers of CNS injury.^6,22,24 NfL and tau have similarly demonstrated strong associations with axonal damage across multiple neurological conditions. ²³ Together, these markers form a biologically complementary panel that captures astroglial, axonal, and neuronal cell body injury.

Furthermore, all four biomarkers are incorporated within the validated Quanterix Simoa Neurology 4-Plex assay, enabling simultaneous ultrasensitive quantification within a standardized multiplex platform. ²⁵ The availability of this well-characterized assay allowed for harmonized measurement with reduced inter-assay variability and facilitated comprehensive evaluation of multiple injury pathways within a single cohort.

The primary aim of this prospective clinical study was to perform a preliminary evaluation of the ability of four brain injury biomarkers: GFAP, NfL, tau, and UCH-L1, to differentiate CT-confirmed ICH patients from healthy controls and to characterize their temporal profiles using the Quanterix 4-plex assay, which employs ultrasensitive paramagnetic enzyme-linked immunosorbent assays in blood in patients who were diagnosed with ICH. In addition to evaluating early diagnostic differentiation, serial sampling through Day 14 was performed to characterize biomarker kinetics after ICH. Consistent with observations in traumatic brain injury, the temporal profile of circulating neuromarkers may provide insight into injury progression, secondary pathophysiological mechanisms, and potential recovery trajectories.^26-28

The secondary aims include 1) comparing blood biomarker concentrations between aged and adult patients diagnosed with ICH and 2) presenting the pilot study results assessing the potential diagnostic value of neuroendocrine hormones, including the adrenocorticotropic hormone (ACTH, pituitary), agouti-related peptide (AgRP, hypothalamic, from NPY-neurons, regulates food intake) ²⁹ ciliary neurotrophic factor (CNTF), a member of the interleukin-6 family of cytokines, is produced following brain injury by astrocytes, ³⁰ and growth hormone (GH, pituitary) are widely researched in other CNS injuries. Our objective was to assess the biomarkers individually rather than integrating them into an established clinical decision-making pathway or guideline.

Materials and Methods

Patient and Clinical Trial Enrollment

The prospective single-center longitudinal cohort study, approved by the Institutional Review Board of the Chinese University of Hong Kong, was conducted between October 2014 and January 2017. Patients were recruited consecutively, meeting the following inclusion criteria: (1) Diagnosis of supratentorial ICH confirmed by cranial CT scan without severe alteration of consciousness; (2) Age ≥ 18 years; (3) Arrival at the hospital within 24 hours after symptom onset; (4) Conservative treatment for hematoma; (5) Voluntary offering of repeated blood samples and compliance with study assessment. Exclusion criteria included: (1) Presence of neurological, neuromuscular, autoimmune diseases, and chronic end-stage diseases; (2) Previous serious systemic illnesses or severe conditions, such as malignant tumors, congenital and acquired coagulation disorders, acute coronary disease, liver cirrhosis, uremia, chronic obstructive pulmonary disease, and ascites; (3) Resuscitated cardiac arrest within 24 hours; (4) Previous history of spinal cord injury (SCI), TBI, or ICH; (5) Hematoma in the subarachnoid cavity or intraventricular space (to insure population homogeneity); (6) Specific conditions such as pregnancy, unavailable samples, and invalid lab results. (7) subjects with incomplete serial sampling (i.e., day 1, 2, 3, 7, 14) were not included in the biomarker analysis. Patients were enrolled consecutively during the study period, and no eligible subjects were selectively excluded or skipped. Participants included in the final analysis were those with complete serial sampling; exclusions were based solely on sample availability rather than clinical characteristics.

Additionally, 30 healthy subjects aged ≥ 18 years with serum samples were recruited as controls. The inclusion criteria for control subjects were as follows: (1) willingness to participate in the study, (2) normal physical examination results, (3) age of ≥ 18 years, and (4) subject normal results in Complete Blood Count (CBC). Exclusion criteria were determined to be the same as those for the ICH group.

For the Pilot study, we evaluated blood ACTH, AgRP, CNTF, and GH levels in 10 control subjects (5 male and 5 female) due to resource and assay limitations and 33 consecutive ICHs, whose day 1 blood sampling was collected between 9 a.m. and 11 a.m. to ensure standardization and reduce the impact of circadian rhythm on plasma hormone levels.

Serum Collection and Processing

Venous blood samples were obtained on days 1 (within 24 hours after symptom onset), 2, 3, 7, and 14. Patients with missing blood samples and invalid lab results were excluded. All assays followed the manufacturer’s protocols. Blood samples were allowed to sit until clot formation, then centrifuged at 1000 rcf for 10 minutes, and the supernatant was distributed into 200 µL aliquots in Eppendorf tubes. These samples were then frozen in an ethanol–dry ice bath and stored at -80°C as de-identified blood samples.

Biochemical Analysis

GFAP, NfL, Tau, and UCH-L1 concentrations were measured using the Simoa® Human Neurology 4-Plex B (N4PB) assay on an SR-X immunoassay analyzer (Quanterix Corp, Boston, Massachusetts, USA). ³¹ ACTH, AgRP, CNTF, and GH concentrations in the peripheral plasma were analyzed on days 1 and 7 with MILLIPLEX MAP Non-Human Primate Pituitary Magnetic Bead Panel 1 - Endocrine Multiplex Assay (NHPPT1MG-46K).

Statistical Analysis

Data was analyzed using IBM SPSS Statistics version 27.0 (IBM Corp., New York City, New York, USA). ³¹ The Kolmogorov-Smirnov Test assessed data homogeneity. Kruskal-Wallis and Mann-Whitney U tests were used for continuous variables if normality was not found. For multiple group comparisons, post-hoc pairwise analyses were performed using Bonferroni correction to account for multiple testing. Data is presented as median (IQR), and categorical variables as numbers and percentages. Receiver operating characteristic (ROC) analysis was performed on day 1(within 24 hours of symptom onset, representing the acute diagnostic phase) and day 7 (representing the early subacute phase) between ICH and healthy control participants to assess their ability to differentiate between ICH patients and healthy controls. A p-value of < 0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****< 0.0001).

Results

The study statistically analyzed seventy-one patients with biomarker results available for all four biomarkers. The ICH patients (n=71) are divided into two subgroups according to their age: (1) the adult group (younger than 60 years; n=47) and the aged group (equal to or above 60 years; n=24) (Table 1).

Table 1.

Demographic Characteristics of Intracerebral Hemorrhage (ICH) Patients and Healthy Controls

​	Age (years) mean ± SD (min-max)	Gender % male
Control (n=30)	39.0±15.2 (18-73)	50.0
Adults (<60 years, n=26)	35.1±12.1 (18-59)	42.3
Aged (≥60 years, n=4)	62.2±5.9 (60-73)	100
ICH (n=71)	55.1±13.8 (18-88)	64.8
Adults (<60 years n=47)	47.4±9.0 (18-58)	65.9
Aged (≥60 years n=24)	70.1±7.8 (60-88)	62.5

Results of Brain-Specific Blood Biomarkers

The results for four biomarker levels in the ICH (n=71) and control group (n=30) are presented in Table 2. The levels of the four biomarkers were statistically significantly higher on all assessed days in patients with the ICH group compared to the control group. The levels of the four biomarkers were significantly higher in ICH patients compared to controls (Supplement Figures 1,2,3,4). ROC curves were used to evaluate the diagnostic value of the four biomarker levels based on the area under the curve for distinguishing healthy controls from day 1 and day 7 ICH, as shown in Figure 1 and Supplement Table 1. GFAP was found to have the most diagnostic brain injury marker characteristics with 100% sensitivity and 100% specificity (Area Under the Curve; ranging from 1.00 to 1.00). Across time points, GFAP showed an early peak followed by a gradual decline, whereas NfL demonstrated progressive elevation over time. Tau and UCH-L1 exhibited early increases with subsequent reduction toward later time points (Table 2).

Table 2.

Results of Brain-Specific Biomarkers: Glial Fibrillary Acidic Protein (GFAP), Neurofilament Light Chain (NfL), Tau, and Ubiquitin C-Terminal Hydrolase-L1 (UCH-L1)

​	GFAP median (pg/ml) (25-75)	NfL median(pg/ml) (25-75)	TAU median(pg/ml) (25-75)	UCH-L1 median(pg/ml) (25-75)
ICH day 1 n=71	9598.4**** (181.5-23586.5)	38.32**** (21.0-68.8)	1.89**** (0.77-3.75)	87.6**** (28.8-174.2)
ICH day 2 n=71	10456.9**** (2097.6-36379.1)	54.01**** (24.4-81.6)	2.81**** (1.0-4.7)	133.65**** (37.5-210.6)
ICH day 3 n=71	5611.8**** (1734.2-22345.2)	74.68**** (38.7-110.6)	2.86**** (1.2-4.9)	106.0**** (38.4-217.4)
ICH day 7 n=71	1234.5**** (619.8-4651.9)	240.1**** (99.2-479.0)	1.20**** (0.7-1.9)	45.5**** (23.5-67.0)
ICH day 14 n=71	568.4**** (267.3-1763.8)	566.7**** (206.8-786.5)	0.73**** (0.4-1.2)	25.7** (16.0-45.4)
Control n=30	39.2 (31.9-50.1)	4.54 (3.7-7.3)	0.20 (0.05-0.4)	16.38 (12.9-22.4)

The Mann–Whitney U Test was Used. ** p<0.01, ****p<0.0001 when compared with the control group.

Figure 1.

Receiver operating characteristic (ROC) curves of glial fibrillary acidic protein (GFAP), neurofilament light chain (NfL), tau, and ubiquitin C-terminal hydrolase-L1 (UCH-L1) on day 1(1A) and day 7 (1B)

The serum concentrations of four biomarkers among adult ICH, aged ICH groups, and the control group are presented in (Supplement Figures 5,6,7,8). A comparison of the three groups using the Kruskal–Walli’s test revealed a statistically significant difference between the three groups (p < 0.05). The Mann–Whitney U test with Bonferroni correction was performed for multiple comparisons. All biomarker levels were significantly higher in the adult ICH and aged ICH groups than in the control group (p<0.0001). However, no significant difference was observed between the adult ICH and aged ICH groups.

Results From the Pilot Study on Neuroendocrine Hormone Alterations After ICH

A total of 43 participants’ results were statistically analyzed in the pilot study. Patients aged 22 to 73 years (mean, 46.6 years; standard deviation, 14.6 years) comprised 20 males and 13 females in the ICH group. In the control group of 10 participants, ages ranged from 52 to 65 years (mean, 57.4 years; standard deviation, 4.1 years), and it consisted of 5 males (60.6%) and 5 females. There were no significant differences between the groups according to age and gender.

The results for ACTH, AgRP, CNTF, and GH levels on only day 1 and day 7 were obtained in blood, and a comparison between the ICH group and control group is presented in Table 3 and Supplement Figures 9,10,11,12. Only the levels of ACTH and AgRP were statistically significantly higher on day 1 and day 7 in patients with the ICH group compared to the control group (p<0.05). ROC curves of ACTH, AgRP, CNTF, and GH on day 1 and day 7 were presented in Figure 2 and Supplement Table 2. To our knowledge, this is the first report describing neuroendocrine AgRP upregulation following ICH.

Table 3.

Results of Neuroendocrine Biomarkers: Adrenocorticotropic Hormone (ACTH), Agouti-Related Peptide (AgRP), Ciliary Neurotrophic Factor (CNTF), and Growth Hormone (GH)

​	ACTH median (pg/ml) (25-75)	AgRP median (pg/ml) (25-75)	CNTF median (pg/ml) (25-75)	GH median (pg/ml) (25-75)
ICH day 1 n=33	34.0**** (26.5-68.0)	190.0**** (123.5-248.0)	16.5 (13.75-21.0)	315.0 (125.7-808.5)
ICH day 7 n=33	41.0**** (26.5-82.5)	177.0**** (111.50-274.25)	16.0 (13.5-18.2)	366.0 (92.5-1220.2)
Control n=10	20.5 (19.7-22.5)	35.5 (20.2-75.0)	12.5 (11.0-24.2)	149.5 (88.0-466.2)

The Mann–Whitney U test was used, ****p<0.0001, when compared with the control group.

Figure 2.

Receiver operating characteristic (ROC) curves of adrenocorticotropic hormone (ACTH), agouti-related peptide (AgRP), ciliary neurotrophic factor (CNTF), and growth hormone (GH) on day 1 (2A) and day 7 (2B)

Discussion

The present study demonstrates a clear differentiation between ICH patients and healthy controls based on four biomarkers in this exploratory cohort, with GFAP demonstrating 100% sensitivity and specificity under the conditions of this study. However, this finding should be interpreted with caution, as the comparison was limited to ICH patients and healthy controls and may not be generalized to clinical practice. There was no statistically significant difference in biomarker concentrations between adult ICH patients under 60 and those aged 60 and above. The relatively younger age of the cohort compared with Western ICH populations may reflect regional epidemiological differences, as s previous studies have reported younger age at stroke onset in Asian populations compared with Western populations.^32,33 When examining ACTH, AgRP, CNTF, and GH, only ACTH and AgRP levels showed significant differences between the control and ICH groups.

The observed performance of GFAP in this study aligns with its established role in TBI, further supporting its potential utility as a blood-based biomarker for acute ICH diagnosis. ²¹ Although not officially recognized as a diagnostic ICH biomarker, our results align with previous studies and meta-analyses demonstrating the potential of plasma GFAP for diagnosing ICH.^14-19,34,35 Notably, Katsanos et al reported significantly higher plasma GFAP values in ICH patients compared to controls, stroke mimics, and acute ischemic stroke when the patients presented to the emergency department within 6 hours from symptom onset. ³⁵ On the other hand, in a study conducted by Rozanski et al of 74 patients, the prehospital GFAP levels in patients with a smaller ICH volume often remained in the normal range. ²⁰ NFL is a major component of neuronal axons and is less investigated in the diagnosis of ICH. Our study findings align with studies showing a rapid response of plasma NfL levels to cerebral insult, suggesting its utility as a temporal biomarker for monitoring axonal injury post-ICH.^12,36

Tau, another well-known biomarker for neurological diseases, including TBI and ischemic stroke, has been explored in the context of ICH. ¹³ We found higher levels of the axonal injury biomarker tau, similar to the study conducted by Tobieson et al in patients with ICH. ¹³

Hu et al investigated the prognostic value of serum tau concentrations in patients with ICH who underwent admission >6 h from symptom onset in the cohort study. ³⁷ However, they did not have a control group and did not evaluate the diagnostic value of Tau in patients with ICH.

UCH-L1 plays a crucial role in synaptic plasticity and homeostasis and contributes to the brain’s intrinsic mechanisms for self-repair following injury. ⁶ While much of the research has focused on UCH-L1 in TBI and neurodegenerative diseases, its role in conditions like stroke and ICH is an emerging area of interest.^10,11,13,38 Similar to our study, serum UCH-L1 was significantly higher in the ICH group compared to the control group in the previous studies.^10,11 In Yigit et al study, UCH-L1 levels were significantly higher in the ICH group compared to controls (p = 0.012), with no correlation between UCH-L1 levels and age, consistent with our findings. ¹⁰

Neuroendocrine alterations have been documented following events such as ischemic stroke, subarachnoid hemorrhage, and TBI. However, to our best knowledge, there is only one article in PubMed regarding ACTH, cortisol, FH, and LH changes in patients diagnosed explicitly with ICH. ³⁹ In one study by Huttner et al, the ACTH level appears to increase from the onset of the event to the third day in patients with ICH, where the hematoma size is less than 20 ml. ³⁹ This result is consistent with our study. However, a decrease in ACTH levels is noteworthy in cases where ICH is greater than 20 cm. In our study, the hematoma level was not assessed with CT. ACTH and cortisol may affect the patient’s hemodynamics; therefore, new studies need to be planned to support the treatment of ICH patients.

AgRP is primarily expressed in the hypothalamus and plays a role in regulating appetite and energy balance. ²⁹ To our knowledge, there is no study on the relationship between AgRP and ICH, TBI, and stroke. Little is known about the expression of AgRP in cases of amyotrophic lateral sclerosis. ²⁹ The present study is the first to show a significant increase in AgRP after ICH. However, it’s important to note that the relationship between AgRP and hypothalamic injury can be complex, and the specific effects may depend on the nature and extent of the injury.

CNTF, an IL-6 family cytokine abundant in the brain, is released from astrocytes after brain injuries and shows increased expression around hematoma lesions and white matter structures, accompanied by astroglia proliferation in ICH rat models. ³⁰ We could not find a clinical study that evaluates the CNTF level after ICH. In the present clinical pilot study, we could not find significant differences between the control group and the ICH group; however, the degree of hemorrhage may affect the result, which was not evaluated in our study.

In this prospective observational pilot study involving 40 consecutive patients with acute ICH, the researchers found that elevated GH levels on admission were associated with increased mortality within 30 days and worse functional outcomes after 90 days. ⁴⁰ In the present study, there were no significant differences between ICH and control patients according to the GH level. However, the relationship may be complex and influenced by various factors, including the severity of the injury, the time elapsed since the event, and individual differences among patients. Further research is needed to fully understand the mechanisms and implications of the ICH-GH relationship.

Our research has several limitations that should be overcome in future studies. First, we did not evaluate the correlation between serum biomarker levels and hematoma location, hematoma volume, and clinical severity scales of patients. Secondly, we don’t have an aged control group to compare with the aged ICH group, which may confound the interpretation of age-sensitive endocrine markers like ACTH and GH. Thirdly, the relatively small sample size limits the scope of our study. In addition, given the evaluation of multiple biomarkers across several time points, the findings should be interpreted as exploratory and require validation in larger cohorts with prespecified statistical strategies. These findings suggest that GFAP, NfL, tau, UCH-L1, ACTH, and AgRP may have potential as diagnostic and monitoring biomarkers in spontaneous ICH; however, these results should be interpreted with caution and require validation in larger studies.

The small sample size in the pilot study also restricts the statistical power to detect meaningful differences for CNTF and GH, which may explain the non-significant findings. Lastly, although blood samples were collected during the early hours following hospital admission, strict control for circadian rhythm was not feasible due to variability in patients’ presentation times. Moreover, potential confounding effects of clinical treatments (e.g., corticosteroids, sedatives) on hormone levels were not systematically assessed, which constitutes a limitation of this study.

In conclusion, our study demonstrates differences in blood-based and neuroendocrine biomarkers between ICH patients and healthy controls in this exploratory cohort. GFAP, NfL, tau, UCH-L1, ACTH, and AgRP showed significant elevations; however, these findings should be interpreted with caution, given the study design, and are not directly generalizable to clinical practice. Further research is needed to establish standardized cut-off values and to evaluate their diagnostic utility in larger, multicenter studies. Additionally, the lack of significant differences between age groups suggests that GFAP, NfL, tau, and UCH-L1 may exhibit consistent patterns across age groups, although this requires further validation.

Limitation

First, the control group was not strictly age-matched to the ICH cohort, which may represent a potential source of confounding because circulating levels of several brain injury biomarkers can vary with age. Age-related variability in blood-based brain biomarkers has been reported in healthy populations, and relevant references have been added to support this observation.^41,42 However, the magnitude of biomarker elevations observed in the ICH cohort was substantially greater than the variations typically attributed to aging. For example, compared with healthy controls, biomarker concentrations in ICH patients showed approximately 246.54-fold higher GFAP levels on Day 1, 124.82-fold higher NfL levels on Day 14, 14.34-fold higher tau levels on Day 3, and 8.24-fold higher UCH-L1 levels on Day 2. Similarly, AgRP levels increased 5.35-fold on Day 1, while ACTH levels increased 2.0-fold on Day 7 relative to controls. These findings indicate a clear separation in biomarker levels between the ICH group and healthy controls; however, they reflect group-level differences and do not establish clinical diagnostic performance.

Nevertheless, confirmation of these findings in larger studies incorporating age-matched control groups will be important to further address this potential source of bias, and this limitation has been further emphasized in the present study. Additionally, the requirement for complete serial sampling may have introduced selection bias, as patients with incomplete sampling were excluded; however, exclusion was based on sample availability rather than predefined clinical characteristics.

Second: the comparison was made between CT-confirmed ICH patients and healthy controls rather than an unselected cohort of patients presenting with suspected acute stroke. Therefore, while the biomarkers demonstrated clear differentiation between confirmed ICH and healthy controls, the present design does not allow for a definitive assessment of clinical diagnostic utility in a real-world stroke triage setting. Evaluation of true diagnostic performance will require prospective studies in unselected suspected stroke populations, with blood sampling obtained prior to imaging confirmation.

Third, detailed clinical severity measures (e.g., NIHSS, GCS), imaging characteristics (hematoma volume and location), treatment variables, and intercurrent complications were not systematically collected within the scope of the original study protocol. As the study has since been formally closed following IRB completion, additional retrospective data extraction was not permissible under institutional regulations. The absence of these clinical and radiographic variables limits correlation analyses between biomarker levels and disease severity or outcomes, and future studies should incorporate comprehensive clinical and imaging characterization.

ORCID iD

Nina Collier https://orcid.org/0009-0002-6943-8142

Ethical Considerations

The prospective single-center longitudinal cohort study, approved by the Institutional Review Board of the Chinese University of Hong Kong/Prince of Wales Hospital, was conducted between October 2014 and January 2017 (Principal Investigator: Wai S. Poon) under protocol #2014-01-01.

Consent to Participate

In accordance with the Declaration of Helsinki (1964), written informed consent was obtained from patients, legal representatives, or their closest relatives.