Accepted Manuscript

Stroke biomarkers in clinical practice: A critical appraisal

Geelyn J.L. Ng, Amy M.L. Quek, Christine Cheung, Thiruma V. Arumugam, Raymond
C.S. Seet

PII: S0197-0186(16)30460-0
DOI: 10.1016/j.neuint.2017.01.005
Reference: NCI 3986

To appear in: Neurochemistry International

Received Date: 21 November 2016

Revised Date: 5 January 2017
Accepted Date: 8 January 2017

Please cite this article as: Ng, G.J.L., Quek, A.M.L., Cheung, C., Arumugam, T.V., Seet, R.C.S., Stroke
biomarkers in clinical practice: A critical appraisal, Neurochemistry International (2017), doi: 10.1016/
j.neuint.2017.01.005.

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 ACCEPTED MANUSCRIPT
STROKE BIOMARKERS IN CLINICAL PRACTICE: A CRITICAL APPRAISAL

Geelyn J.L. Ng a,b, Amy M.L. Quek a,b, Christine Cheung c,d, Thiruma V. Arumugam e,

Raymond C.S. Seet a,b

a
Division of Neurology, Department of Medicine, Yong Loo Lin School of Medicine,

National University of Singapore, Singapore

b
Division of Neurology, Department of Medicine, National University Health System,

Singapore

c
Institute of Molecular and Cell Biology, Agency for Science, Technology and

Research, Singapore

d
Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore

e
Department of Physiology, Yong Loo Lin School of Medicine, National University of

Singapore, Singapore

Corresponding author:

Dr Raymond Chee Seong Seet

Division of Neurology, Department of Medicine

Yong Loo Lin School of Medicine, National University of Singapore

NUHS Tower Block Level 10, 1E Kent Ridge Road

Singapore 119228

Keywords: Ischemic stroke; biomarkers; oxidative damage; inflammation;

etiology; hemorrhagic transformation

Running Title: Stroke biomarkers in clinical practice

1
ABSTRACT

Biomarkers provide critical mechanistic insights to key biologic processes that occur

during cerebral ischemia which, when carefully applied, can improve clinical

decision-making in acute stroke management. The translation of a blood-based

biomarker in ischemic stroke to clinical practice is challenging, in part, due to the

complexity of ischemic stroke pathogenesis and the presence of a blood-brain

barrier that restricts the release of brain-specific markers into the circulation. The

pathologic and clinical aspects of ischemic stroke are described in this review, where

a non-exhaustive list of biomarkers that interrogate different aspects of ischemic

stroke such as oxidative damage, inflammation, thrombus formation, cardiac function

and brain injury are described. The potential roles of these biomarkers are further

examined under different clinical scenarios aimed at (1) averting the risk of

hemorrhagic transformation, (2) identifying individuals at risk of early neurologic

deterioration and malignant infarction, (3) aiding in the diagnosis of ischemic stroke

and its differentiation from other stroke mimics, (4) guiding the search for stroke

etiology, and (5) assessing stroke risk within the community. Researchers should

explore the roles of stroke biomarkers to enhance clinical decision-making that is

presently largely based on intuition and subjective reasoning.

INTRODUCTION

By 2050, more than 1.5 billion people in the world will be aged 65 years and older,

and a silent epidemic of stroke is imminent 1. Stroke represents the second leading

cause of death for people older than 60 years, the most important cause of

permanent disability, and uses approximately 3-7% of the total healthcare

expenditure in high-income countries1.

The pathophysiology of cerebral ischemia has played a key role in guiding

biomarker research in ischemic stroke. Substantial data indicate that atherosclerosis

is a life course disease that begins insidiously with the evolution of risk factors, giving

rise to a subtle subclinical disease that culminates in overt cerebrovascular

illnesses2,3. Ischemic stroke is a consequence of atherosclerosis that affects the

large intra- and extracranial arteries and small vessels, as well as a result of an

embolic phenomenon of blood thrombus from the heart or aorta, resulting in an

interruption and severe reduction of blood flow within the cerebral circulation 4.

Depending on the degree of hypoperfusion, an area with complete absence of flow

can result, namely the infarct core, where neuronal death occurs within a few

minutes, and a surrounding area called the penumbra which suffers from a moderate

reduction of blood flow and contains functionally impaired but semi-viable brain

tissues5. In the ischemic core region, brain cells undergo necrotic cell death

producing an area that is electrically, metabolically and functionally inactive. By

contrast, neurons in the ischemic penumbra are thought to be metabolically active,

but electrically and functionally compromised. The penumbra has a variable

outcome. If blood flow is not restored within a relatively short time, the penumbra

undergoes the same destiny as the core region 4.

 Central to the acute treatment of ischemic stroke are arterial recanalization

and reperfusion by means of intravenous recombinant tissue plasminogen activator

(TPA)6 and endovascular treatment (through device-driven retrieval or aspiration of

blood thrombus)7. Early reperfusion can potentially salvage ischemic brain tissues

and, in clinical trials, is associated with a 5-fold increase in the likelihood of a good

functional recovery7. Upon reoxygenation, reactive oxygen species (ROS) and early

inflammatory cells are rapidly recruited and numerous non-enzymatic oxidation

reactions take place in the cytosol and/or cellular organelles 8. The ischemic cascade

is characterized by the following biochemical events - bioenergetics failure, ionic

imbalance, acidosis, excitotoxicity, oxidative stress and inflammation, before

culminating in cell death via necrosis or apoptosis 8. In practice, the benefits of

arterial recanalization and reperfusion are weighed against the dreaded risk of

intracranial hemorrhage that is associated with early neurologic deterioration,

malignant infarction and a high mortality9.

The ability to identify high-risk stroke patients is desirable for clinicians to

accurately triage patients to specialized stroke units for closer monitoring,

individualize treatment in anticipation of stroke-related complications, and accurately

inform long-term prognosis. Current methods to identify such high-risk individuals

depend largely on clinical intuition that is derived from an assessment of neurologic

and neuroimaging features, and initial treatment response. The use of biological

signatures of cerebral ischemia that takes into account the complex biology of stroke

is appealing to clinicians as this facilitates an objective assessment of benefits and

risks under different clinical scenarios.

This review provides an overview of the mechanistic basis of a non-

exhaustive list of biomarkers that interrogate different aspects of stroke pathogenesis

(e.g. oxidative damage, inflammation, thrombus formation, cardiac function and brain

injury) and describes their potential applications to guide clinical decision-making at

critical time-points in stroke management. This review focuses on biomarkers that

are measurable in blood, a material that is widely accessible in human stroke. We

searched medical databases such as MEDLINE, PubMed and Ovid for publications

that highlight the use of blood-based biomarkers in clinical scenarios where

comparisons between biomarkers are made with outcomes such as hemorrhagic

transformation, early neurologic deterioration and malignant infarction.

WHAT IS A BIOMARKER?

Biomarkers are objectively-measured biological signatures of normal and pathologic

processes that can serve a wide range of purposes such as risk stratification,

therapeutic assessment strategies, clinical trial design and drug development 10.

Simply, a biomarker has good clinical acceptance if the biomarker is accurate, is

acceptable to the patient, is easy to interpret by clinicians, has a high sensitivity and

specificity for the outcome it is expected to identify, and explains a reasonable

proportion of the outcome independent of established predictors consistently across

multiple studies. Biomarkers also provide an avenue for researchers to gain a

mechanistic understanding of the differences in pharmacological responses to drugs

in preclinical and human models, and improve the design of clinical trials by

tightening the selection criteria to better target the application of a particular

compound of interest11.

As clinical events take time to develop (sometimes years), the use of

biomarkers as surrogate endpoints is appealing to clinical trialists and

epidemiologists as this might shorten the duration of study and reduce the overall
cost of screening the efficacy of a compound of interest. Several features of a good

surrogate biomarker include a biomarker that tracks closely with changes in the

outcome of interest and one that is validated in independent cohorts 12,13. In a

heterogeneous disease such as stroke, the use of multiple biomarkers may be

necessary to reflect the different facades of complex disease pathways and yield

surrogate endpoints that offer a more comprehensive assessment of treatment

effects. The source of biomarkers is equally as important. In stroke patients, a

biomarker that is measured in human brain tissues is impracticable as such tissues

are often inaccessible; neither is it possible to routinely obtain cerebrospinal fluid

from stroke patients who are receiving either antiplatelet or anticoagulation treatment

that puts them at an increased risk of bleeding following lumbar puncture. In patients

with stroke mimics such as intracerebral hemorrhage and tumor, performing a

lumbar puncture may trigger cerebral herniation especially among patients with

undiagnosed intracranial hypertension. Among the elderly with degenerative disc

disease, lumbar puncture can be technically difficult to perform which might result in

an unacceptably long delay to obtain an adequate sample for acute clinical decision-

making. In normal individuals, brain-derived biomarkers are not typically detectable

in large amounts in the circulation due to the integrity of the blood-brain barrier

(BBB). By contrast, acute disruptions in the BBB following ischemic stroke can

trigger the release of brain-derived biomarkers into the circulation, thereby providing

a novel window into the pathophysiology of stroke for wider applications of brain-

derived biomarkers in clinical stroke management.

TYPE OF BIOMARKERS

Following cerebral ischemia, an increase in ROS occurs, triggering an activation of

downstream inflammatory responses, where recruitment of multiple immune cells

such as macrophages, neutrophils and T cells to the damaged brain tissues

occurs14. These inflammatory cells release pro-inflammatory cytokines such as

interleukin-6 (IL-6) that are capable of crossing the BBB into the circulation 15.

Another class of proteins that is also involved in the inflammatory response of stroke

is the Toll-like receptors (TLRs)16. In addition, biomarkers such as D-dimer and those

that play a part in platelet function have been implicated in thrombus formation and

propagation. A major source of thrombus is the heart and large vessels such as the

aorta where failure of the cardiovascular system to deliver sufficient blood to the

brain can exacerbate stroke outcomes. Patients with disorders in cardiac rhythm

such as atrial fibrillation (AF) have been shown to harbor increased stroke risk 17.

Examples of cardiac biomarkers are the N-terminal pro-B-type natriuretic peptide

(NT-proBNP)18 and high sensitivity troponin T (hsTNT) 19. Due to the heterogeneity

and multiple pathways implicated in stroke, the selection of biomarkers presented in

this review covers key biological processes in stroke pathogenesis and is not

intended to be exhaustive.

Biomarkers of oxidative damage

Oxidative damage results from an imbalance in redox state where the pro-oxidant

effects overwhelm the antioxidant capacity of brain tissues. The burden of oxidative

damage accumulates in individuals with vascular risk factors such as diabetes

mellitus, cigarette smoking, advanced age and hypertension, and predisposes these

individuals to cardiovascular complications such as stroke and myocardial

infarction20. The brain, in particular, is highly susceptible to oxidative damage owing

to its high consumption of oxygen, rich iron content, high concentrations of readily

peroxidizable lipids, and relatively low endogenous antioxidant capacity 21. Following

cerebral ischemia, ROS triggers disruption in cellular structural integrity and

functions through lipid peroxidation, DNA fragmentation, and protein

denaturation22,23. Severe cellular injury occurs, leading to cerebral infarction and, not

infrequently, infarct expansion 24. Although several reliable biomarkers of oxidative

damage exist, the roles of two notable biomarkers of oxidative damage for stroke are

reviewed here, namely uric acid and F2-isoprostanes.

Uric acid. Uric acid, which exists almost entirely in its ionized form urate at

physiological pH, is derived from adenine- and guanine-based purine compounds 25.

The ability of uric acid to scavenge peroxynitrite, hydroxyl radical and singlet oxygen,

and chelate transition metals, suggests an antioxidant role of urate in humans25. Uric

acid is degraded in most mammals by the hepatic enzyme, urate oxidase (uricase),

to allantoin. In humans, however, mutations in early hominoids that rendered the

uricase gene non-functional have resulted in urate levels in human plasma that are

approximately 10 times those of most other mammals 26. In vivo, uric acid is a potent

water-soluble antioxidant that targets free radicals caused by oxidative damage,

including hydroxyl radicals and superoxide 27,28 but, at higher levels, could exhibit

paradoxical pro-oxidant effects 29. Data supporting the neuroprotective functions of

uric acid in ischemic stroke are largely borne out of observational and interventional

trials. In a prospective study involving 881 consecutive patients, uric acid levels were

inversely associated with the extent of neurological deficits on admission and the

final infarct volume on CT/MRI scans 30. In patients receiving intravenous

thrombolysis, uric acid levels correlated with better neurological outcomes, whereas
patients with malignant middle cerebral artery infarction and symptomatic intracranial

hemorrhage have significantly lower uric acid levels31. The value of replenishing uric

acid in the latter group of patients has been investigated by trials that examined the

dual administration of uric acid and TPA during acute ischemic stroke 32. Despite

early promising findings, in the definitive URICO-ICTUS phase 2b/3 trial that

recruited 411 patients with acute ischemic stroke, the addition of uric acid to

thrombolytic therapy did not increase the proportion of patients who achieved

excellent outcome after stroke compared with placebo 33.

F2-Isoprostanes. F2-Isoprostanes are a family of prostaglandin isomers that are

formed as a result of non-enzymatic peroxidation of arachidonic acid induced by free

radicals20,34-38. F2-Isoprostanes are widely considered as accurate and reliable

biomarkers of oxidative damage that can be measured in plasma, serum, urine,

saliva and cerebrospinal fluid39. F2-isoprostanes are measured in nanomolar units

and are accurately measured using analytical platforms such as high performance

liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS)

and light chromatography-mass spectrometry (LC-MS). F 2-isoprostanes have been

studied among individuals with various neurological conditions such as Parkinson

disease35 and among individuals with vascular risk factors such diabetes mellitus 40,

hypertension41 and cigarette smoking36.

Despite a mounting evidence implicating F 2-isoprostanes in human diseases,

few studies have examined F2-isoprostanes in human stroke. Sanchez-Moreno and

colleagues first reported higher F 2-isoprostanes levels within 5 days following

stroke42. Ward and colleagues supported these findings and similarly showed an

increase in F2-isoprostanes levels during the early course of ischemic stroke 43. Our

group subsequently showed that the rise in F2-isoprostanes occurred as early as

three hours after ischemic stroke onset and remained elevated for several days 20. In

the Biomarker Evaluation for Antioxidant Therapies in Stroke (BEAT-Stroke) study,

Kelly and colleagues observed that early F 2-isoprostanes levels within 8 hours

following the onset of ischemic stroke correlated with MMP-9 levels 44. Taken

together, these data suggest that F 2-isoprostanes are sensitive markers of oxidative

damage in human stroke that are elevated early and remain elevated for several

days.

Biomarkers of inflammation

In the intact brain, trafficking of cellular and molecular components from the

peripheral circulation is regulated by the BBB. However, after an ischemic brain

injury, the tight junctions between endothelial cells of the BBB become leaky, which

allow circulatory immune cells to permeate and infiltrate the surrounding brain

parenchyma. These immune cells secrete pro-inflammatory cytokines (such as IL-6

and TLRs) to overcome the injurious damage following stroke.

Interleukin-6. A majority of inflammatory reactions to acute cerebral ischemia are

mediated by cytokines that increase in the central nervous system (CNS) and the

systemic circulation in patients with acute ischemic stroke. Glycoprotein interleukin-6

(IL-6) is a crucial inflammatory cytokine that is released in the post-stroke setting and

serves as a vital messenger molecule between leucocytes, vascular endothelium,

and parenchyma resident cells. IL-6 has pleomorphic effects in the brain resulting in

anti-apoptotic, pro-proliferative, growth-inhibitory, and differentiation-inducing effects

in various diseases45,46. Although an over-expression of IL-6 by astrocytes and

microglia might have dual roles in acute ischemia (with both neurotoxic and

neuroprotective effects attributed to this cytokine), blood levels of IL-6 have been
repeatedly associated with poor outcome after stroke 47. IL-6 concentration in blood

has been correlated with baseline stroke severity, suggesting a plausible role as a

biomarker of acute cerebral injury 48. In the clinical setting, IL-6 has been measured

following the onset of acute ischemic stroke, consistently showing an increase in IL-6

levels46. In some studies, an increase in IL-6 correlated with a corresponding rise in

C-reactive protein (CRP)46,49.

Toll-like Receptors. Toll-like receptors (TLRs) belong to a family of transmembrane

pattern recognition receptors (PRRs) that recognize highly preserved structures

known as pathogen-associated molecular patterns (PAMPs) in pathogens and

damage-associated molecular patterns (DAMPs) from endogenous molecules 50,51.

When cerebral ischemia sets in, TLRs recognize DAMP signals that are released as

a result of tissue damage. These molecules, also known as endogenous ligands, are

able to activate TLRs and trigger an inflammatory response 16,52. TLR activation

promotes the recruitment of several adaptive proteins to activate nuclear factor-κB

which, in turn, induces the expression of pro-inflammatory genes, inflammatory

cytokines including IL-6, and adhesion molecules 53.

Among members of the TLR family, TLR2, TLR4, TLR8 and TLR9 have been

closely associated with stroke events. In separate studies, the expressions of TLR2

and TLR4 were associated with poor outcomes in ischemic stroke patients 54,55.

Ischemic stroke induces the release of DAMPs, such as cellular fibronectin (c-Fn),

HSP60, and HSP70, which function as endogenous ligands for TLR2 and TLR4, and

promoting activation of the inflammatory cascade 52. TLR8 activation plays a

detrimental role in stroke outcome by promoting neuronal apoptosis and T cell-

mediated post-stroke inflammation 56, although this has only been demonstrated in

mice and clinical evidence is, at present, still lacking.

Biomarkers of thrombus formation

Platelets play a crucial role in the pathogenesis of atherosclerosis, thrombosis, and

stroke. Thrombus formation (also known as thrombogenesis) results from abnormal

adherence and aggregation of platelets to the vessel wall, leading to an interruption

of blood flow into the brain. In a thromboembolic stroke, blood thrombus that is

formed from large arteries of the aorta or the heart can be dislodged into the cerebral

circulation. Aspirin, used alone or in combination with other antiplatelet drugs, has

been shown to confer significant benefit to patients at high risk of vascular events.

Resistance to the action of aspirin can occur and this might decrease its benefit to

mitigate against stroke risks. The definitions of aspirin resistance vary considerably

across studies depending on the type of assays used. Despite the importance of

aspirin resistance in clinical practice, the heterogeneity in the definitions of aspirin

resistance has led to a lack of consensus on the roles of platelet function testing in

stroke management.

D-Dimers. Plasma D-dimer is a fibrinogen degradation product that reflects thrombin

production and fibrinolysis. In the general population, D-dimer levels vary and tend to

correlate positively with other cardiovascular risk factors 57, where the presence of D-

dimer confirms the generation of thrombin and plasmin 58. As fibrin D-dimer is

considered to originate from cross-linked fibrin assembled during thrombus

formation, a rise in fibrin D-dimer levels indicates thrombus formation. While there

are other markers for thrombus formation such as prothrombin fragments 1+2

(F1+2), thrombin-antithrombin complexes (TAT), D-dimer has been demonstrated to

be most stable as compared to the other thrombotic markers following ischemic

stroke59.
Platelet Reactivity. Platelets have been shown to contribute to the pathogenesis

and/or propagation of cerebral infarction by forming occlusive aggregates. Platelet

inhibition is important, not only in the chronic phase but also in the acute phase of

brain ischemia, to prevent platelet aggregation and early vascular recurrence 60.

Antiplatelet agents such as aspirin and clopidogrel have been shown to reduce the

risk of recurrent vascular events and death in stroke patients 61. The ability to identify

patients who are resistant to aspirin or clopidogrel early during their course of stroke

is appealing as this can lead to a selection of a suitable antiplatelet agent for

secondary prevention. The concept of antiplatelet resistance is borne out of

observations that certain patients do not derive protection from antiplatelet treatment.

For example, aspirin resistance can be identified by measuring thromboxane A 2

production and platelet aggregation 61, or measuring urinary levels of 11-

dehydrxythrombxane B262. The value of platelet function testing is not clear in stroke

patients given the relative scarcity of data in this area. It remains unclear whether

changing a therapy in patients identified to be aspirin resistant actually results in

improved outcomes. One study suggested that in stroke patients with a level of

monocyte chemotactic protein-1 (MCP-1) >217 pg/ml, outcomes were better at 90

days in those treated with aspirin plus extended release dipyridamole (Aggrenox)

compared with aspirin alone63. High uric acid levels has been shown to predict

aspirin resistance where a cut-off value of 6.45 mg/dl of uric acid predicted aspirin

resistance with 79% sensitivity and 65% 64. These findings suggest a potential

application of platelet biomarkers to personalize the choice of antiplatelet treatment

for cardiovascular disease prevention.

Biomarkers of Cardiac Function

The brain depends on the heart to effectively pump and deliver sufficient blood to the

cerebrovascular circulation. Interruptions in blood flow to the heart-brain axis could

result in a state of cerebral hypoperfusion and promote development of blood

thrombus within the cardiac atrium and left atrial appendage. A widely recognized

cardiac risk factor of stroke is AF that results in a chaotic firing of electrical activities,

causing stasis of blood within the heart and, in the presence of thrombogenic factors,

predispose to thrombus formation. Several cardiac biomarkers that have increasingly

been studied include the brain natriuretic peptide (BNP) and high-sensitivity troponin

T (hsTnT).

N-terminal Pro-Brain Natriuretic Peptide. Brain natriuretic peptide (BNP) is a

neurohormone that is produced by the heart ventricles and the brain 65,66. BNP

promotes natriuresis and diuresis in the body, acting as a vasodilator with countering

vasoconstrictor effects67,68. After the cleavage of a propeptide, two fragments are

formed - BNP, which is biologically active, and N-terminal proBNP (NT-proBNP),

which does not have biological activity. Both of these biomarkers are present in

identical concentrations among normal individuals69. BNP has been widely studied in

ischemic stroke patients; a recent pooled data meta-analysis concluded NT-proBNP

fragment levels to be useful in identifying patients with cardioembolic stroke 70.

High Sensitivity Troponin T. Troponin is a highly sensitive and specific marker of

myocardial necrosis that is used in the diagnosis of acute myocardial infarction and

to stratify patients according to the severity of their acute coronary symptoms 19.

Some studies have reported an association between elevated levels of cardiac

troponin T and poor outcomes in acute stroke71-73. In a meta-analysis, an elevation in

cardiac troponin levels in stroke patients was associated with an overall increased

risk of death and disability19.

Biomarkers of Brain Injury

S100β. S100β is a glial protein that belongs to the S100 family that regulates

intracellular calcium. The homodimeric S100β protein consists of two β subunits that

are largely expressed by astrocytes and, to a lesser extent, by neurons, microglia,

and oligodendrocytes74,75. By modulating secondary messenger calcium signaling,

S100β participates in cellular differentiation and motility 76. S100β is protective and

trophic at low concentrations, but can also be toxic and pro-apoptotic at high levels.

When structural damage such as infarction occurs, S100β is released into the

cerebrospinal fluid and, depending on the integrity of the blood-brain barrier, is

detectable in blood. In normal individuals, S100β concentration is approximately 40

times higher in cerebrospinal fluid than in serum. Previous studies by Aurell et al.

and Büttner et al. have demonstrated that the glial protein S100β was increased in

ischemic stroke patients who present 1 to 7 days after the insult 77,78.

Neuron-specific enolase. Neuron-specific enolase (NSE) is a cytosolic brain enzyme

that functions as a glycolytic isoenzyme79,80. NSE is confined solely in neurons under

normal conditions and only a negligible amount of NSE is physiologically present in

blood81. NSE is released in the systemic circulation after ischemic stroke; therefore,

an increase in NSE levels in the peripheral blood can be taken as an indicator of the

integrity of the BBB and infarct volume.

APPLICATIONS OF BLOOD-BASED BIOMARKERS IN THE CLINICAL SETTING

Biomarkers can be classified by their intended clinical application. In ischemic stroke,

studies have evaluated biomarkers to distinguish ischemic stroke from its mimics,

determine stroke etiology, predict stroke severity and outcomes (e.g. early

neurological deterioration and hemorrhagic complications), and identify patients who

might benefit from decompressive hemicranietomy. Several studies have also

examined the use of biomarkers to prognosticate patients for outcomes such as

functional recovery and vascular events (e.g. recurrent stroke, myocardial infarction

and death)82. Several researchers have investigated biomarkers that estimate infarct

volume and have found significant associations between infarct volume and

biomarkers such as S100β, matrix metallopeptidase-9 (MMP-9), IL-6, tumor necrosis

factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1) and glutamate 44,48,83.

Such findings, however, have limited clinical application as physicians depend

largely on a detailed neurological examination and interpretation of neuroimaging

findings to ascertain stroke severity 80. Furthermore, stroke volume itself may not be

a reliable indicator of stroke severity as a small stroke may not be any less

innocuous especially when critical or eloquent areas of the brain such as the

brainstem or language areas are affected. By contrast, large areas of injury may

cause subtle clinical manifestations (e.g. strokes affecting the non-dominant

temporal lobe). Nonetheless, predicting an outcome elusive to clinical and

neuroimaging findings, based on a blood sample acquired at the time of stroke

presentation, could have practical value in the clinical setting (e.g. risk of

hemorrhagic transformation, early neurologic deterioration and malignant infarction).

Hemorrhagic transformation. When assessing stroke patients for reperfusion

treatment, the beneficial effects of TPA to achieve arterial recanalization are often

balanced against the risk of symptomatic intracranial hemorrhage (SICH). Early

recognition of patients at high risk for SICH is important to triage these patients into

specialized stroke units for closer monitoring and tighter control of risk factors

(especially blood pressure), and a reassessment of their suitability for intravenous

thrombolysis. As disruptions in the BBB have been implicated in hemorrhagic

transformation, current research has focused on biomarkers indicating early BBB as

a predictor for hemorrhagic transformation. To date, the roles of biomarker have

been explored through the measurement of MMP-9 which in vivo is involved in

destruction of microvascular integrity by degradation of the basal lamina and

extracellular matrix84. TPA itself activates MMP-9 that is capable of disrupting BBB

and hemorrhagic transformation85,86. Before the administration of intravenous

recombinant TPA, serum MMP-9 levels >140 ng/ml predicted the development of

intracranial hemorrhage in ischemic stroke patients 87.

Cellular fibronectin (c-Fn) is another factor that has been associated with

increased hemorrhagic transformation. c-Fn is synthesized by endothelial cells and

is elevated following ischemic stroke. A study showed c-Fn levels >3.6 µg/ml predict

the development of hemorrhagic transformation following TPA use 88. Other

biomarkers that have been evaluated to predict the development of intracranial

hemorrhage include plasminogen activator inhibitor type 1 (PAI-1) and thrombin-

activated fibrinolysis inhibitor (TAFI) 89,90. A study of 77 ischemic stroke patients

identified lower levels of PAI-1 and TAFI to be associated with hemorrhagic

transformation91. In combination, PAI-1 levels >180% and TAFI levels <21.4 ng/ml

predicted SICH after TPA treatment89.

Early neurologic deterioration. Early neurological deterioration (END), defined as

worsening of neurological status from admission to 48-72 hours after admission,

affects up to 40% of stroke patients 92,93. END occurs due to the development of

complications following stroke such as failure of collateral circulation, thrombus

progression, recurrent stroke, brain edema or herniation, and hemorrhagic

transformation. Although existing management of END targets the prevention of

these complications, early recognition of END and admission of stroke patients into

specialized stroke units have been demonstrated to reduce the rate of poor

outcome94. As stroke care facilities are scarce, an accurate identification of patients

is central to the cost-effective use of these resources. Biomarkers may have an

important role in this setting to identify these high-risk patients, especially since

biomarkers such as glutamate, gamma-Aminobutyric acid (GABA), ferritin, TNF-α,

ICAM-1, and MMPs have been associated with END 95-97. For example, plasma

glutamate levels of >200 µmol/l predicted END in patients with hemispheric stroke 96

and correlated with infarct growth determined using diffusion-weighted imaging 89.

High-sensitivity CRP, a systematic marker of inflammation, has been found

alongside high-density lipoprotein (HDL) and the presence of internal carotid artery

occlusion to be associated with END98. Recently, plasma levels of soluble form of

receptor for advanced glycation end products (sRAGE) have been correlated with 3-

month functional recovery post-stroke 99; it would be of interest to compare these

levels with END.

Malignant infarction. Patients with an ischemic stroke affecting the anterior

circulation (e.g. internal carotid and middle cerebral artery occlusion) are at risk of

developing space-occupying edema and cerebral herniation that is life-

threatening100. A randomized multicenter study revealed a benefit for early

decompressive surgery for patients with “malignant infarctions” to divert raised

intracranial pressure away from the brainstem101. In one study, plasma S100β level

>0.35 g/l predicted the development of malignant infarction at 12h with a 75%

sensitivity and 80% specificity, and at 24h with a 94% sensitivity and 83% specificity.

In another study, c-Fn and MMP-9 levels were found to be higher in patients with

malignant infarctions. c-Fn >16.6 µg/ml had a sensitivity of 90% and a specificity of

100% for the prediction of a malignant course of infarction 97. Trial data on the use of

these biomarkers to guide clinical decisions leading to early decompressive surgery

are currently lacking.

Stroke diagnosis. Patients experiencing a sudden-onset, focal neurological deficit

may have a stroke or they may have one of several possible “stroke mimic”

conditions, including the aura of migraine, postictal deficits following a focal seizure,

metabolic derangements (especially hypoglycaemia), recrudescence of prior deficits

in the setting of an infection or metabolic derangement, mass lesions or a functional

psychogenic spell. In prospective series, stroke mimics can account for up to a third

of patients presenting with stroke-like symptoms 102. Although there may be clinical

accompaniments to help distinguish between these possibilities (e.g. typical migraine

preceding neurologic symptoms, jerking movements and sphincteric disturbances

following a seizure), these features are often absent and occur unwitnessed. As a

significant proportion of patients have altered consciousness and speech abilities,

this information is not often conveyed to clinicians who make time-sensitive

decisions on acute reperfusion treatment.

Although advanced neuroimaging methods (e.g. magnetic resonance

imaging, vascular and perfusion scans) could aid in the differentiation of stroke from

its mimics103, these investigations take time to organize even in comprehensive

stroke centers. In many centers, however, these neuroimaging facilities are not

available. It is plausible that a single or a panel of diagnostic stroke biomarkers could

complement CT scan findings by confirming the presence of brain-derived

biomarkers in the circulation. In the setting of pre-hospital stroke care, these

diagnostic stroke biomarkers could be used to triage patients for neuroprotection

agents to either cease or slow down the detrimental cascade of events triggered by

the ischemic insult before a definitive reperfusion treatment is administered. Several

studies have reported the ability of single and multiple panel biomarkers to

distinguish stroke from controls with high sensitivity and specificity by targeting

antibodies to the glutamate NMDA-R (NR2A/NR2B subunits) 83,104 and an NMDA-R

peptide105. Reynolds and colleagues had previously screened plasma samples from

223 stroke patients (including ischemic stroke, intracerebral hemorrhage and

subarachnoid hemorrhage) and from 214 healthy individuals for more than 50 serum

biomarkers106. Univariate analysis revealed astroglial protein S100β, B-type

neurotrophic growth factor, von Willebrand factor (vWF), MMP-9 and monocyte

chemotactic protein-1 (MCP-1) to be associated with stroke. The same group

published data on 65 patients with suspected ischemic stroke admitted within 24

hours of symptom onset and 157 healthy controls 107. Twenty-six blood-based

markers with relation to the ischemic cascade were analyzed. Protein S100β, MMP-

9, vascular cell adhesion molecule (VCAM-1), and vWF were identified to be

associated with ischemic stroke. Subsequently, Laskowitz and colleagues obtained

data on 130 patients admitted with acute focal neurologic deficits within 6 hours of

symptom onset108. Forty-one patients were later on diagnosed with ischemic stroke.

The predictive model included BNP, CRP, D-dimer, MMP-9, and S100β.
 Based on these encouraging results, the diagnostic accuracy of a biomarker

panel including D-dimer, BNP, MMP-9, and S100β was evaluated in a prospective

multicenter trial109. Within a 3-year period, more than 1,100 patients presenting with

symptoms suspicious for stroke were enrolled within 24 hours of symptom onset.

The multivariate model was capable of only moderately differentiating between

stroke patients and mimics. Setting the threshold of the model to the 25th percentile

revealed a sensitivity of 86% and a specificity of 37% for discriminating stroke

patients from mimics, which is of limited use to guide decision-making relating to

thrombolysis. The use of a single or a panel of biomarkers as an adjunct to

neuroimaging-guided triaging of patients to exclude stroke mimics remains

unexplored.

Stroke etiology. Several etiologies of stroke exist (cardioembolism, large artery

disease and lacunar stroke) and determining the underlying cause of stroke is

paramount when planning for secondary prevention treatment. For example, patients

with a cardioembolic stroke benefit substantially from anticoagulation, whereas

treatment strategies for patients with a large artery disease and lacunar stroke center

around antiplatelet treatment and strict risk factor control 110. Additionally, patients

with a symptomatic large artery disease due to carotid stenosis could benefit from

either endarterectomy or stenting. Despite an extensive search for stroke etiology,

the etiology of stroke remains unknown in up to 35% of patients111,112. The prospects

of employing a biomarker-based platform to elucidate on the cause of stroke are

appealing as this might direct the search for stroke etiology and rationalize the need

for expensive investigations (e.g. angiographic studies, echocardiogram and

prolonged cardiac rhythm monitoring). Both the pro-BNP and D-dimer have been

shown to reliably distinguish cardioembolic from non-cardioembolic strokes113,114,115.

Pooled data from 23 studies that include clinical information from 2,834 patients

showed significant elevation in pro-BNP levels in cardioembolic stroke within 72

hours from symptom onset70. Conversely, RNA expression in blood has been shown

to distinguish cardioembolic stroke from large artery disease. In a study of 194

samples from 76 patients with acute ischemic stroke, a 40-gene panel was able to

distinguish strokes from a cardioembolic from large artery disease etiology with

>95% sensitivity and specificity at 3, 5 and 24 hours after stroke onset116.

In patients with cryptogenic stroke, identification of patients at risk for the

occurrence of AF is important to decide on the need and the duration of prolonged

cardiac monitoring117. Current methods of patient selection are largely based on

loose clinical criteria such as age, the pattern of stroke on neuroimaging studies and

the number of cardiovascular risk factors 117,118. A study by Rodriguez-Yanez and

colleagues demonstrated the value of measuring pro-BNP where pro-BNP ≥360

pg/ml was the only variable that independently associated with the risk of developing

119
AF .

Stroke risk. Several biomarkers have been shown to predict the risk of recurrent

ischemic events following ischemic stroke. Lipoprotein-associated phospholipase A 2

(Lp-PLA2) is associated with a 2-fold increase in stroke occurrence and risk of

recurrent stroke120,121 and high-sensitivity CRP levels have also been shown to

predict the risk of incident stroke122. High levels of oxidized low-density lipoprotein

(oxLDL), a biomarker closely linked with atherosclerosis, has recently been shown to

be associated with one-year mortality and poor functional outcome, especially

among patients with large artery and lacunar stroke mechanisms 123. Conversely,

high-density lipoproteins (HDLs) have been observed to be anti-atherosclerotic 124,

anti-thrombotic125, anti-inflammatory126, as well as antioxidant127; hence, it may also
be an effective biomarker for stroke treatment. Another notable example is peptide

midregional proadrenomedullin (MR-proADM), a plasma biomarker which has been

demonstrated to be elevated in ischemic stroke patients with poor functional

recovery128.

Endothelial markers such as dimethylarginines (which in vivo are endogenous

inhibitors of nitric oxide synthase) have been associated with atherosclerosis and

stroke risk factors. A longitudinal study involving 67 ischemic stroke patients has

demonstrated significant elevations in asymmetric dimethylarginine (ADMA) and

symmetric dimethylarginine (SDMA) following stroke; the extent of increase within 72

hours from stroke onset predicted poor functional recovery 129.

AF is the commonest sustained cardiac rhythm disorder that has been

associated with an increased mortality and morbidity from stroke and

thromboembolism17. Using data from the ARISTOTLE and STABILITY trials, Hijazi

and colleagues developed a risk assessment tool that is based on multiple

biomarkers including NT-proBNP (indicating myocyte stress) and cardiac hsTnT

(indicating myocardial injury) to assign AF-associated stroke risk. The ABC-stroke

(Age, Biomarkers, Clinical history of stroke or transient ischemic attack [TIA]) yielded

higher C-indices than the widely used CHA 2DS2-VASc scores to guide selection of

high-risk patients for long-term anticoagulation 130. In a study of 880 patients with

non-valvular AF, CRP levels positively correlated with the CHADS 2 stroke risk

assessment scores and were higher in patients with all-cause mortality and vascular

events. However, no significant correlation was found with stroke risk. 131 Among

individuals with high cardiovascular risk, our group has shown significant elevations

of multiple biomarkers of oxidative damage such as F 2-isoprostanes,

neuroprostanes, 7α-hydroxycholesterol and γ-glutamyltransferase41. In

anticoagulated non-valvular AF patients, urinary F 2-isoprostanes predicted

cardiovascular events and all-cause mortality, and the addition of tertiles of urinary

F2-isoprostanes to CHA2DS2-VASc improved the performance of each outcome 132.

CONCLUSIONS

Blood-based biomarkers provide useful insights to the pathological events leading to

cerebral infarction and could add to the armamentarium of clinical tools in stroke

management. Although the ischemic cascade has been extensively studied in animal

stroke models, few studies have examined the prognostic significance and temporal

release of circulatory biomarkers that examine the different states of oxidative

damage, inflammation, hemostasis, neuronal/glial injury and cardiac dysfunction in

human stroke. More efforts should focus on the standardization of study cohorts and

analytical methods, incremental yield of different markers and cost-effectiveness of

incorporating biomarker measurement to guide clinical decision-making. Rather than

pursuing a search for an elusive troponin-equivalent biomarker, researchers should

consider focusing their efforts to contextualize and applying existing biomarkers to

clinical scenarios where decision-making is still largely based on intuition and

subjective reasoning. Given the wide stroke heterogeneity, the use of single

biomarkers may not sufficiently capture the different aspects of stroke pathogenesis;

instead, multiple biomarkers that assess the significance of various stroke-related

molecular alterations may be necessary. The translation of stroke biomarkers to

clinical practice is challenging but can be extremely rewarding, especially when such

concerted efforts of researchers, clinicians, industry partners and regulatory

authorities result in a positive outcome for stroke patients.

ACKNOWLEDGEMENTS

We would like to thank the National Medical Research Council, Singapore

(NMRC/CSA-SI/0003/2015, NMRC/CNIG/1115/2014 and

NMRC/MOHIAFCat1/0015/2014) for their generous support.

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Table 1. Biomarkers that assess different aspects of stroke pathogenesis

Type Biomarker Description References

21,25-33,40,42,64,82,
Purine metabolism product; has both
Uric acid 93
antioxidant and pro-oxidant properties
Oxidative
damage
F2- Prostaglandin isomers, free radical 20,21,34-44,132
isoprostanes product of arachidonic acid

Inflammatory cytokine, messenger 15,16,45-48,50-52,

molecule between leucocytes, the 55-57,80,97,98,107,
IL-6 vascular endothelium, and parenchyma
116,131
resident cells

An acute-phase pentameric protein 10,18-20,35,40-42,

found in the blood plasma; a type of 44-46,48,49,55,57,
CRP pattern recognition receptors (PRR) 59,64,73,82,98,108,
involved in inflammation response and
Inflammation innate immunity 116,120-122,131

A family of transmembrane PRRs that

recognize highly preserved structures,
pathogen-associated molecular 16,50-56,116
TLR
patterns (PAMPs) in pathogens and
damage-associated molecular patterns
(DAMPS), from endogenous molecules

Fibrinogen degradation product that

57-59,73,82,104,108,
reflects thrombin production and
D-dimer 109,113-115
fibrinolysis; presence also confirms
Thrombus generation of plasmin
formation
Forms occlusive aggregates that
Platelet 60-64
propagate cerebral infarction; inhibition
Reactivity presents vascular recurrence

18,65-70,73,82,108,
Neurohormone that promotes
NT-proBNP natriuresis and diuresis in the body 109,113-115,119,130

Cardiac
function Marker of myocardial necrosis; used to
19,71-73,80,82,106,
diagnose acute myocardial infarction
hsTnT and prognostic markers of ischemic 107,109,130
stroke

Homodimeric glial protein that regulates

intracellular calcium levels, also a 74-79,82,89,106-109
Brain Injury S100β
marker of blood-brain barrier
dysfunction
 Dimeric isoenzyme, predominantly
79-82
NSE found in neurons and neuroendocrine
cells

Abbreviations: IL-6, interleukin-6; CRP, C-reactive protein; TLR, toll-like receptor; NT-proBNP, N-
terminal pro-brain natriuretic peptide; hsTNT, high sensitivity troponin T; NSE, neuron-specific
enolase.
Table 2. Application of stroke biomarkers under different clinical scenarios

Type Biomarker Description References

Calcium-dependent proteolytic enzyme,

8,16,44,52,84-88,97,
involved in degradation of basal lamina
MMPs 106-109,116
and extracellular matrix to disrupt blood-
brain barrier

Major component of extracellular matrix,

52,82,88,97
c-Fn involved in wound healing and cell
Hemorrhagic adhesion
Transformation
Protease inhibitor, inhibits tissue 89-91
PAI-1 plasminogen activator (tPA)

Enzyme that hydrolyzes fibrin C-terminal

89-91
TAFI bonds to prevent fibrinolysis and inhibits
activation of plasminogen

Amino acid; neurotransmitter that 4,38,82,83,89,96,97,

Glutamate activates cell surface receptors found on 104,105
neuronal and glial membranes

Inhibitory neurotransmitter that reduces

95,97
GABA neuronal excitability by binding to
transmembrane receptors on neurons

Intracellular protein involved in storing

Ferritin and transporting iron 21,82,96

Cell signalling protein that regulates

15,16,42,47,51,52,54,

Early TNF-α 55,80,84,97,116

immune cells and inhibits tumorigenesis
Neurologic
Deterioration
Transmembrane immunoglobulin protein
42,48,52,80
ICAM-1 involved in leukocyte-endothelial cell
signal transduction

Calcium-dependent proteolytic enzyme,

8,16,44,52,84-88,97,
involved in degradation of basal lamina
MMPs 106-109,116
and extracellular matrix to disrupt blood-
brain barrier

An acute-phase pentameric protein 10,18-20,35,40-42,

found in the blood plasma; a type of 44-46,48,49,55,57,
CRP pattern recognition receptors (PRR) 59,64,73,82,98,108,
involved in inflammation response and
116,120-122,131
innate immunity
 ACCEPTED MANUSCRIPT

Homodimeric glial protein that regulates

74-79,82,89,106-109
S100β intracellular calcium levels, also a
marker of blood-brain barrier dysfunction

Major component of extracellular matrix,

Malignant 52,82,88,97
c-Fn involved in wound healing and cell
Infarction adhesion

Calcium-dependent proteolytic enzyme,

8,16,44,52,84-88,97,
involved in degradation of basal lamina
MMPs 106-109,116
and extracellular matrix to disrupt blood-
brain barrier

Amino acid; neurotransmitter that 4,38,82,83,89,96,97,

Glutamate activates cell surface receptors found on 104,105
neuronal and glial membranes

Homodimeric glial protein that regulates

74-79,82,89,106-109
S100β intracellular calcium levels, also a
marker of blood-brain barrier dysfunction

Plasma glycoprotein involved in platelet

vWF adhesion 59,106,107

Calcium-dependent proteolytic enzyme,

8,16,44,52,84-88,97,
MMPs involved in degradation of basal lamina
and extracellular matrix to disrupt blood- 106-109,116
brain barrier

Chemokine that regulates migration and

42,63,106
MCP-1 infiltration of monocytes and
Stroke macrophages
Diagnosis
Transmembrane immunoglobulin protein
52,107
VCAM-1 involved in leukocyte-endothelial cell
signal transduction

18,65-70,73,82,108,
Neurohormone that promotes natriuresis
NT-proBNP 109,113-115,119,130
and diuresis in the body

An acute-phase pentameric protein 10,18-20,35,40-42,

found in the blood plasma; a type of 44-46,48,49,55,57,
CRP pattern recognition receptors (PRR) 59,64,73,82,98,108,
involved in inflammation response and
116,120-122,131
innate immunity

Fibrinogen degradation product that

57-59,73,82,104,108,
reflects thrombin production and
D-dimer 109,113-115
fibrinolysis; presence also confirms
generation of plasmin

37
 18,65-70,73,82,108,
Neurohormone that promotes natriuresis
NT-proBNP 109,113-115,119,130
and diuresis in the body

Stroke Etiology
Fibrinogen degradation product that
57-59,73,82,104,108,
reflects thrombin production and
D-dimer 109,113-115
fibrinolysis; presence also confirms
generation of plasmin

Phospholipase A2 enzyme that catalyzes

35,40,82,120-122
Lp-PLA2 the degradation of platelet-activating
factor by hydrolysis

Oxidative modification of low-density

123
oxLDL lipoprotein that is involved in the
development of atherosclerotic plaques.

A major group of lipoprotein that has

been shown to be anti-atherosclerotic, 124-127
HDL
anti-thrombotic, anti-inflammatory and
antioxidant.

128
MR-proADM A vasodilatory peptide found in plasma

Endogenous inhibitor of nitric oxide

129
ADMA synthase; released by endothelial cells
and causes endothelial dysfunction
Stroke Risk
129
SDMA Structural isomer of ADMA

CRP
An acute-phase pentameric protein 10,18-20,35,40-42,
found in the blood plasma; a type of 44-46,48,49,55,57,
pattern recognition receptors (PRR) 59,64,73,82,98,108,
involved in inflammation response and
116,120-122,131
innate immunity

18,65-70,73,82,108,
Neurohormone that promotes natriuresis
NT-proBNP 109,113-115,119,130
and diuresis in the body

F2- Marker of
myocardial
hsTnT isoprostanes
necrosis; used to
diagnose acute
myocardial infarction and
prognostic markers of ischemic
19,71-73,80,82,106,
stroke
107,109,130
Prostaglandin isomers, free radical
product of arachidonic acid
20,21,34-44,132
 ACCEPTED MANUSCRIPT
Abbreviations: MMPs, matrix metalloproteinases; c-Fn, cellular fibronectin; PAI-1, plasminogen
activator inhibitor type 1; TAFI, thrombin-activated fibrolysis inhibitor; GABA, gamma-Aminobutyric
acid; TNF-α, tumor necrosis factor alpha; ICAM-1, intercellular adhesion molecule 1; CRP, C-reactive
protein; vWF, von Willebrand factor; MCP-1, monocyte chemotactic protein-1; VCAM-1, vascular cell
adhesion protein 1; NT-proBNP, N-terminal pro-brain natriuretic peptide; Lp-PLA 2, lipoprotein-
associated phospholipase A2; oxLDL, oxidized low-density lipoprotein; HDL, high-density lipoprotein;
MR-proADM, midregional proadrenomedullin; ADMA, asymmetric dimethylarginine; SDMA, symmetric
dimethylarginine; hsTNT, high sensitivity troponin T.

40
 ACCEPTED MANUSCRIPT

 Biomarkers provide mechanistic insights to key biologic processes during stroke.

 Biomarkers can aid in clinical decision-making during stroke management.

 Prognostic significance of stroke biomarkers could be further examined.