Liquid biopsy markers for stroke diagnosis

Abstract

Introduction:

There is a short time window (4.5 h) for the effective treatment of acute ischemic stroke (AIS), which uses recombinant tissue plasminogen activator (rt-PA). Unfortunately, this short therapeutic timeframe is a contributing factor to the relatively small number of patients (~7%) that receive rt-PA. While neuroimaging is the major diagnostic for AIS, more timely decisions could be made using a molecular diagnostic.

Areas Covered:

In this review, we survey neuroimaging techniques used to diagnose stroke and their limitations. We also highlight the potential of various molecular/cellular biomarkers, especially peripheral blood-based (i.e., liquid biopsy) biomarkers, for diagnosing stroke to allow for precision decisions on managing stroke in a timely manner. Both protein and nucleic acid molecular biomarkers are reviewed. In particular, mRNA markers are discussed for AIS and hemorrhagic stroke diagnosis sourced from both cells and extracellular vesicles.

Expert Opinion:

While there are a plethora of molecular markers for stroke diagnosis that have been reported, they have yet to be FDA-cleared. Possible reasons include the inability for these markers to appear in sufficient quantities for highly sensitive clinical decisions within the rt-PA therapeutic time

1.0. Introduction

1.1. Stroke and stroke statistics.

Stroke is defined as a syndrome of brain dysfunction with an underlying vascular cause. According to the American Stroke Association and the CDC in 2019, stroke was a leading cause of serious long-term disability and the 5^th leading cause of death in the United States [1]. Every year, more than 795,000 people in the United States have a stroke with 185,000 patients experiencing a recurrent event [2]. The costs of stroke associated with health care services, treatment, and missed work productivity in the US alone is estimated at $34 billion each year [3].

Medical conditions such as high blood pressure, high cholesterol, heart disease, diabetes, and obesity can increase stroke risk. Sociodemographic and geographic disparities persist where the incidence of stroke is significantly higher. The geographical distribution of stroke events in the US shows a “Stroke Belt”, an area in the Southeastern US with the highest rate of stroke mortality. Place of residence likely contributes to the development of stroke risk factors such as norms influencing dietary patterns, exposure to life stressors, access to healthcare, and lifestyle choices including lack of regular physical activity and the highest prevalence of cardiovascular risk factors [7].

The problem of stroke is not limited to the US; in 2015 stroke was responsible for 11.8% of total deaths worldwide, which makes stroke the second leading global cause of death behind heart disease. Although the overall risk of stroke has dropped by about 25% during the last decade [8], disability caused by strokes has become one of the major health problems worldwide. Therefore, there is a need to improve healthcare facilities for stroke rehabilitation and the early detection of stroke conditions to predict the recurrence of the disease and for the primary prevention of stroke.

1.2. Areas covered.

The literature selected for this review surveys neuroimaging techniques used to diagnose stroke currently along their benefits and limitations. Works reviewed include molecular and cellular biomarkers, especially those isolated from peripheral blood that can serve as a liquid biopsy for stroke diagnostics or to deliver prognostic and predictive information to allow for the implementation of precision decisions on managing acute stroke.

2.0. Stroke types

The American Stroke Association has identified two major types of stroke, namely acute ischemic stroke, AIS (blockage of blood supply to part of the brain), and hemorrhagic stroke (a rupture of the blood vessel in the brain). Clearly, the functional changes caused by both of these subtypes are different. Thus, it is important that the underlying mechanisms involved in these stroke types are understood so that the diagnosis and prognosis can be delineated.

In terms of epidemiology for AIS and intracerebral hemorrhage (ICH), older age and hypertension are major risk factors for both stroke types. Diabetes, atrial fibrillation, previous myocardial infarction, previous stroke, and intermittent arterial claudication are common risk factors for AIS, while high alcohol consumption is more commonly associated with hemorrhagic stroke. Although AIS and ICH cannot be reliably distinguished on clinical grounds, ICH tends to be more severe than AIS, and within the first 1–3 months is associated with a considerable increase of mortality, which is specifically associated with the hemorrhagic nature of the lesion.

2.1. AIS.

AIS is the most common type of stroke, which accounts for approximately ~87% of all strokes (Table 1). AIS occurs when a clot or a mass clogs a blood vessel that supplies blood to the brain (Figure 1A). This obstructs blood flow to brain cells and when this happens, brain cells become deprived of oxygen [9]. During an AIS, brain damage and neuronal death occur due to the failure of energy producing compounds, such as adenosine triphosphate (ATP), which in turn results in reduced glucose production and limits oxygen to the brain. As a result, cellular homeostasis is not supported. Due to the lack of energy, functioning of the ion gradient is also affected by the loss of ions, such as potassium resulting in neuronal swelling. Other complex mechanisms are involved in brain tissue affected by AIS, such as triggering of the release of glutamate and aspartate neurotransmitters in the brain, dysfunction of the calcium channel, and production of reactive oxygen species that can activate proteases and lipases that can damage cellular and extracellular elements. As a final result of these processes, immediate death of a section of the brain parenchyma (core) or partial injury (penumbra) with the potential recovery with fast treatment can take place [10]. Brain damage caused by AIS is determined by many factors including duration, severity, and location of the event.

Table 1.

Sub-types of stroke.

Stroke Types
Ischemic (85% – 87%)		Hemorrhagic (13% – 15%)
Embolic (~35%)	Thrombotic (~52%)	Intracerebral (~10%)	Subarachnoid (~5%)

Figure 1.

(A) Representation of a blood clot in a blood vessel that supplies blood to the brain (reprinted from [4]). (B) Representation of hemorrhagic stroke; subarachnoid hemorrhage and intracerebral hemorrhage (reprinted from [5]). (C) Summary of common stroke mimics and the frequency of hteir appearance as identified in a systematic review (reprinted with permission from [6]. Copyright 2014 Korean Stroke Society).

The main cause for AIS is fatty deposits on vessel walls called atherosclerosis. Cerebral thrombosis is a thrombus or a blood clot that will develop overlying the fatty plaque within the blood vessel while cerebral embolism is a blood clot that forms at another location in the circulatory system (Table 1). These are usually formed in heart and large arteries of the upper chest and neck. These blood clots break loose, enter the bloodstream, and move to the brain’s vessels until they reach one that is too small to let it pass creating an obstruction for blood flow. Irregular heartbeat, which is called atrial fibrillation, is one of main reasons of embolism that causes these clots to form in the heart [11]. Additional causes of AIS are small vessel disease and less frequent etiologies such as arterial dissection, other vasculopathies, cardiac diseases (e.g., mechanical heart valve with superimposed thrombus) and hypercoagulable disorders. Symptoms for AIS and ICH include trouble with speaking and understanding, experience of confusion, and slur of words. Also, sudden numbness, weakness, or paralysis in the face, legs or arms can develop, which usually takes place in only one side of the body and face. Patients may also have blurred or blackened vision in one or sometimes both eyes.

2.2. Hemorrhagic stroke.

Approximately 13–15% of all strokes are hemorrhagic stroke. This stroke type is caused when a weakened blood vessel ruptures causing leakage of blood into the surrounding brain. Blood accumulation and compression of the surrounding brain tissue can damage the brain cells prohibiting proper function (Figure 1B). There are two major types of hemorrhagic stroke namely, intracerebral hemorrhage and subarachnoid hemorrhage (Table 1). In the first type, the bleeding takes place inside of the brain, which is also the main type of hemorrhagic stroke. Risk factors for intracerebral hemorrhage (ICH) include hypertension, which is the most common cause. Other causes of ICH are cerebral amyloid angiopathy, anticoagulation use, cerebral cavernous malfunctions that occurs when blood vessels do not form correctly in the brain, and arteriovenous malformations (AVM), which is a genetic condition where blood vessels form incorrectly resulting in an abnormal vascular tangle.

In subarachnoid hemorrhage, the bleeding will occur between the brain and the membranes that cover it. Risk factors for subarachnoid hemorrhage are a bulge in a blood vessel wall (i.e., aneurysm), AVM, bleeding, head injury, or trauma. Symptoms of ICH may change, but the common signs are headache, loss of balance and inability to coordinate body movement, vision changes, numbness in an arm or leg, seizures, altered consciousness, confusion or loss of alertness, nausea and vomiting [12]. Symptoms of subarachnoid hemorrhage are sudden severe headache, typically described as the worst headache of the patient’s life, vomiting, neck stiffness and sudden collapse with unconsciousness.

Regardless of the type of stroke, patients experiencing signs of stroke can be described by the acronym “FAST”. Face: if you ask the person to smile, one side of the face will drop. Arms: when you ask the person to raise both arms, one arm will drift down or not raise at all. Speech: the person will be unable to repeat a simple phrase or their speech will be slurred or strange, and Time: if you see any of these signs it is indication to seek immediate medical attention [13]. Unfortunately, due to the lack of the awareness of stroke symptoms, some patients with stroke do not arrive to the hospitals on time to receive proper treatment.

2.3. Transient ischemic attacks (TIA).

TIAs are called mini-strokes and are a neurologic dysfunction caused by a loss of blood flow to the brain or spinal cord without acute infarction. Because the blockage period in TIA is short, there is no permanent brain damage. However, in patients who had a TIA the risk of occurrence of a following stroke increases by ~11% over the next 7 days and 24–29% within 5 y [14]. Considering that TIA can lead to recurrent stroke conditions, differentiating between stroke or TIA versus so called stroke mimics is necessary.

2.3. Stroke mimics (SMs).

SMs have similar symptoms as stroke and are often diagnosed as stroke, however, are not stroke. SMs are expressed as diseases caused by neurologic symptoms that resemble a stroke [15]. One of the most common SMs is seizure that often causes neuronal dysfunction manifested by weakness, aphasia, confusion or other sensory signs affecting vision and speech. Complex migraines are another SM that can result in hemiparalysis, vision loss, aphasia, or vertigo [16]. Other SMs are demyelinating disease, meningitis, glucose level variations and metabolic disorders (hypoglycemia), tumors, non-cerebrovascular diseases such as epilepsy, and dementia [17]. SMs that are frequently misdiagnosed as stroke are summarized in Figure 1C [18].

3.0. Stroke assessment scales

Stroke type needs to be diagnosed quickly and precisely so that treatment can be delivered rapidly. The longer the treatment for stroke is delayed, the less effective its results; thus the phrase “time lost is brain lost”. Quick and accurate diagnosis of stroke subtypes eliminating SMs is highly important to provide proper treatment for stroke patients.

Medical personnel evaluates the severity of the stroke using established criteria. Stroke scales are divided into the following categories: (i) Glasgow outcome scale (GOS) used to assess disability; (ii) Glasgow Coma Scale (GCS) to evaluate the severity of an acute brain injury; (iii) physical deficit scale (so called National Institutes of Health Stroke Scale (NIHSS)), that is based on the scores given during neurological assessment of the patient; and (iv) activities of daily living scale (i.e., Barthel index), that is based on the functional outcome and recovery of the patient and useful during rehabilitation [19]. The diagnostic accuracy of these scales is ~80%, however, the clinical specificity and sensitivity vary [19].

Table 2 explains the scale used by the National Institute of Health Stroke Scale (NIHSS), which is considered to be the most widely used scale that can be completed within 5–8 min and yields a numerical score from 0–42 (i.e., higher the score, more severe the stroke). Both, neurologists and non-neurologists can use the test to assess the patient’s conditions. Unfortunately, scales alone cannot be used to distinguish stroke subtypes (i.e., AIS vs. hemorrhagic). These tests are used only to understand the severity of the stroke and are beneficial when used together with brain imaging techniques and biomarkers for diagnosing particular stroke conditions.

Table 2.

National Institutes of Health Stroke Scale (NIHSS) [20].

Question	Response Score
1a. Level of consciousness	0= alert, 1=not alert, 2 = obtunded, 3 = unresponsive
1b. Level of consciousness questions	0 = answers both questions correctly, 1= answers one question correctly, 2 = answers neither correctly
1b. Level of consciousness commands	0 = performs both tasks correctly, 1 = performs one task correctly 2 = performs neither task correctly
2. Gaze	0 = normal, 1 = partial gaze palsy, 2 = total gaze palsy
3. Visual fields	0 = no visual loss, 1= partial hemianopsia, 2 = complete hemianopsia, 3 = bilateral hemianopsia
4. Facial palsy	0 = normal, 1 = minor paralysis, 2 = partial paralysis, 3 = complete paralysis
5. Motor arm  (a) Left  (b) Right	0 = no drift, 1 = drifts before 5s, 2 = falls before 10s, 3 = no effort against gravity, 4 = no movement
6. Motor leg  (a) Left   (b) Right	0 = no drift, 1= drifts before 5s, 2 = falls before 10s, 3 = no effort against gravity, 4 = no movement
7. Ataxia	0 = Absent, 1 = 1 limb, 2 = 2 limbs
8. Sensory	0 = normal, 1= mild loss, 2 = severe loss
9. Language	0 = normal, 1 = mild aphasia, 2 = severe aphasia, 3 = mute or global aphasia
10. Dysarthria	0 = normal, 1= mild, 2 = severe
11. Extinction/inattention	0 = normal, 1= mild, 2 = severe

4.0. Stroke diagnostic methods: neuroimaging

Diagnosing the subtype of stroke quickly and precisely is important to decide proper treatment for patients. The most commonly used stroke diagnostic methods available today are neuroimaging techniques. Imaging methods serve two main purposes. First, to rule out SMs and identify the type of stroke and second to assess the location of the stroke in the brain and the damage caused by the stroke. Currently available neuroimaging techniques and their advantages and disadvantages are briefly discussed below.

4.1. Computed Tomography (CT).

A CT of the brain is a non-invasive diagnostic method that uses X-rays to produce images of the brain. Imaging using CT relies on the same principle as regular X-rays, hence, the use of high radiation doses in all CT imaging techniques, which can be viewed as a disadvantage. X-rays are absorbed differently by different parts of the body. Bones will absorb most of the X-rays, therefore, the skull will appear white in color in the image. Water in the cerebral ventricles or fluid-filled cavities in the middle of the brain absorbs less X-rays and those areas appear black. The brain has intermediate density, hence will appear grey in the CT image. Most AIS causing obstructions are less dense than normal brain, thus appear darker. During a CT scan, the X-ray beam will move in a circle around the body allowing different views of the brain. The X-ray data are displayed as a two-dimensional (2D) image. There are several types of CT-based methods, such as non-contrast CT (NCCT), CT angiogram (CTA), and perfusion CT (PCT); Figure 2A–D [21].

Figure 2.

Demonstration of multimodal CT acquisition, including: (A) Non-contrast CT; (B) CT angiography; and (C) perfusion CT. The patient was a 91-year-old man who was presented with acute onset of slurred speech (reprinted from [21], Copyright 2011, The American Society for Experimental NeuroTherapeutics). (D) CT angiography of the brain showing an occlusion in the distal part of the left M1 segment of the middle cerebral artery (arrow). Reprinted from [22], Copyright 2016; published by S. Karger AG, Basel.

4.2. Non-contrast Computed Tomography (NCCT).

NCCT is widely used to identify early signs of stroke conditions and to rule out hemorrhagic stroke and tumor lesions. Its wide availability and the speed of image acquisition makes it effective for initial evaluation of suspected stroke patients [23]. Advantages of NCCT include rapid access, availability in an emergency departments 24 h and evidence that its use is cost effective [24]. However, there are drawbacks. NCCT scans do not detect chronic hemorrhage including micro-bleeds. Also, NCCT performs poorly in detecting acute infarctions, particularly in posterior fossa due to beam hardening artifacts and insufficient contrast resolution. Acute posterior ischemia stroke accounts for ~20% of AIS [25]. In a recent study, the sensitivity for detecting AIS using NCCT varied from 57% to 71% [26]. Early signs of acute infarctions are very subtle, especially for the smaller arterial blockings and in the hyper acute stage of AIS, which result in limited sensitivity and reduced inter-observer reliability [27]. Furthermore, a study reported that within 6 h after onset of stroke symptoms (i.e., past the timeframe of the administration of thrombolytic therapy), inter-observer agreement (median of 30 CT scans and six raters) of all early infarctions was 61 ±21% with the inter-observer agreement ranging between 14% and 78% for any early infarction. In the same study, it was reported that the mean sensitivity and specificity for detection of early infarctions with CT were 66% (range 20–87%) and 87% (range 56%−100%), respectively [28]. According to the literature, the sensitivity of standard non-contrast CT for AIS increases after about 24 h [29].

Another challenge for neuroimaging techniques are SMs. SMs account for 19–30% of all suspected stroke presentations. Differentiation of SMs from AIS is challenging, given a narrow time window for the administration of intravenous thrombolysis treatment and the required differential diagnoses of stroke to manage patients at the point of referral [18].

4.3. Computed Tomography Angiography (CTA).

In CTA, an iodinated contrast agent is injected intravenously and time-optimized scanning is activated (Figure 2B, D) [30]. This method is widely used to examine blood vessels in the brain. The literature demonstrates some unique benefits of CTA for AIS diagnosis. CTA may facilitate improved pre-procedure planning, and allow for quick treatment decisions. Compared to CT images, CTA images provide higher resolution. When AIS cannot be detected by NCCT, the blockage can be visible in CTA (Figure 2D) [22].

While CTA outperforms NCCT, potential drawbacks of CTA exist. The iodinated contrast agent can potentially initiate an allergic or toxic response in some patients. Thus, if the patient is >60 y of age or if kidney disease, diabetes, lupus, or multiple myeloma were diagnosed in a patient of any age, a time consuming blood test must be performed beforehand to make sure that the contrast agent is safe. Moreover, CTA imaging depends on good timing of measurements following agent administration, technical planning, and sufficient cardiac output [30]. If these conditions are not met, CTA will yield poor images with insufficient diagnostic information. In addition, a CTA can be performed only once every 48 h.

4.4. Computed Tomography Perfusion (CTP).

CTP requires a rapid injection of intravenous contrast agent and repeated imaging of the brain, based on the total amount (Cerebral Blood volume, CBV) and the speed that blood flows (Mean Transient Time, MTT) to different areas of the brain. CTP can help in evaluating the potential areas of salvageable tissue in ischemic penumbra defined as the area of the brain that has reduced blood flow, but increased blood volume. Figure 2C shows a CTP scan of a patient with acute onset AIS.

A recent study suggested that CTP increases the diagnostic accuracy during early stage stroke detection and it can lead to an increased diagnostic yield compared to CT scans (80% vs. 50%, respectively) [31]. The drawbacks of CTP are: The use of CTP requires a higher level of expertise and resources compared to CT or CTA scans; and evaluating the images needs a high level of skill [31]. Additionally, CTP images are spatially limited to 2–4 consecutive sections with coverage of 20–40 mm thickness; this can underestimate the full extent of brain perfusion [30].

4.5. Magnetic Resonance Imaging (MRI).

MRI provides improved sensitivity compared to CT scans with the advantage of avoiding exposure to ionizing radiation and iodinated contrast agents. MRI is a non-invasive test based on a magnetic field altering the alignment of protons in the body, including the brain. When a radiofrequency current is pulsed through the patient’s body, protons are stimulated, and spin out of equilibrium straining against the pull of the magnetic field. When the radiofrequency field is turned off, the MRI detector is able to detect the energy released as the protons realign with the magnetic field [32]. Important MRI techniques used in stroke diagnosis are gradient echo, fluid-attenuated inversion recovery imaging, MR angiography, diffusion-weighted imaging, and perfusion weighted imaging.

4.6. Gradient echo (GRE) and Fluid-attenuated inversion recovery imaging (FLAIR).

GRE is sensitive for detecting acute hemorrhage and micro-bleeds that cannot be detected by NCCT. FLAIR imaging can detect subtle subarachnoid hemorrhages, which can also be undetectable through CT imaging [23].

4.7. Magnetic Resonance Angiography (MRA).

MRA is used for evaluation of large, proximal arteries. The sensitivity and the specificity of MRA in detection of cervical and intracranial stenosis is reported to be in the range of 70% - 100% [8]. Time-of-flight MRA is another method that is used to assess intracranial vasculature. Many studies have been conducted to compare MRA and CTA for visualization of occlusion and stenosis of major cerebral vessels. One study showed that CTA had higher sensitivity for both stenosis and occlusion of major vessels (98% and 100%, respectively) than MRA (70% and 87%, respectively) [33].

4.8. Diffusion Weighted Imaging (DWI).

DWI is a form of MRI based on measuring the random Brownian motion of water molecules within a tissue. The net effect of water that is moving from extra- to intra-cellular space is reduced water mobility due to intra-cellular structural and molecular components acting as barriers of free motion (i.e., cellular swelling exhibit lower water diffusion). This phenomena is captured as hyper-intensity on DWI and hypo-intensity on apparent diffusion coefficient (ADC) maps [25].

DWI is considered to be the most reliable method for the early detection of cerebral ischemia and for detection of many SMs with a reported sensitivity and specificity of 81–100% and 86–100%, respectively [34,35]. DWI can produce false positive and false negative results; non-ischemic lesions including demyelinating diseases, can cause neurologic symptoms and reduced perfusion and lead to mistakenly detected infarcts. If DWI is used together with conventional MRI like FLAIR, it’s possible to differentiate these conditions from infarcts [35].

MRI techniques compared to CT provide better ability to identify both types of strokes. However, the major disadvantage is that MRIs are not always available under emergency conditions. In a study conducted on availability and quality of MRI equipment in US emergency settings, the authors reported that in the US in 2008, only 13% of hospitals had 24/7 MRI services and only 26% of hospitals had 24/7 on-call technologists. [37].

A recent study [36] examined 1689 hospitals of which 961 were certified as stroke centers and 728 were late adopters (Figure 3A). The study found that these centers (certified in a voluntary program) were established unevenly across geographic locations in the US (Figure 3B). As of 2017, stroke-certified hospitals predominantly appear in high-income areas, which can have implications for population-level disparities of stroke care.

Figure 3.

(A) Number of annually stroke-certified hospitals in the US. (B) Stroke-certified hospital locations and hospital service area income distribution. Reprinted from [36], an open access article.

The advantages and limitations of each of the presented imaging technique are summarized in Table 3. As can be seen, due to limitations of these methods, patients with stroke are unable to reach proper treatment during the limited time window for effective treatment of AIS using rt-PA. Hence, there is an urgent need to find new biomarkers that are capable of detecting stroke conditions within a shorter time interval. Currently, there is no FDA-approved molecular diagnostic test for both types of stroke.

Table 3.

Advantages and limitation of neuroimaging techniques. Adapted from [38].

I Imaging Features for Stroke Diagnostics	CT	MRI
Availability in the acute setting (0–6 hours)	++	−
Rapid image acquisition	++	+
Differentiation between acute and chronic ischemia	−	++
Detection of chronic hemorrhage including micro-bleeds	−	+
Sensitivity to lacunarand posterior fossa infarcts	−	++
Differentiation between acute and chronic ischemia	−	++
Ability to assess causes of subarachnoid and intracerebral hemorrhage whille in the scanner	+	+
Time for post-processing angiography and perfusión imaging	−	−
Accessibility for patients with monitors and Ventilators	++	−
Lack of vulnerability to motion artifacts	+	−
Feasibility and safety for patients with metallic implants (pacemakers, implantable defibrillators)	++	−
Lack of ionizing radiation	−	++
Renal toxicity associated with contrast administration	+	+
Cost	+	++

(−) none/low,(+) medium, (++) high

5.0. Therapeutics for stroke

Following a stroke event, there is a low or limited blood supply to the area of the brain affected by stroke. Thus, neurons in these areas are at risk of dying with elapsing time causing detrimental effects on the patients’ brain function [21]. This is the reason why the time between stroke symptoms, diagnosis, and therapeutic treatment is essential and typically represents a relatively short time window. In the next sections, therapeutic approaches available for ischemic and hemorrhagic strokes are detailed.

5.1. Therapeutic approaches for AIS.

Tissue plasminogen activator (t-PA) is a serine protease that cleaves peptide bonds in proteins. It consists of 527 amino acids with 3 or 4 glycosylation sites and 17 disulfide bonds [39]. Vascular endothelial cells are the main source of plasma t-PA that is involved in the breakdown of blood clots (i.e., fibrinolysis), which is the major physiological function of t-PA in blood. With the aid of recombinant biotechnology, t-PA is manufactured in laboratories and this synthetic product is called recombinant tissue plasminogen activator (rt-PA). rt-PA is the only FDA approved drug that is currently available for AIS. Although the proportion of patients with AIS who are treated with rt-PA has increased since its first approval in 1996, the treatment rate is still low and only about 7% of all patients having AIS in the US receive treatment [40]. Reasons for this low rate are due to delays in the emergency medical services, medical contradictions, lack of imaging equipment, and the narrow therapeutic time window for rt-PA treatment [41]. In addition, time-consuming imaging tests contribute to this low treatment rate as well.

The major limitation in using rt-PA is that per regulatory approval, it can be administered only within a window of up to 4.5 h from the onset of stroke symptoms. Although, studies have been conducted toward increasing this effective time window, results have shown that increasing the time length from 4–6 h made rt-PA treatment less beneficial [42]. Under certain conditions, rt-PA used at times beyond the 4.5 h time window can induce brain hemorrhage and injury [43]. Many complex mechanisms have been proposed for hemorrhagic transformation including tPA-mediated N-methyl-D-aspartate excitotoxicity and tPA-mediated microglial inflammation [44]. Research performed using experimental models has suggested the involvement of the extracellular protease family of matrix metalloproteinases (MMPs) that degrade basal lamina and blood-brain barrier substrates, which eventually lead to edema and vascular rupture [45]. Potential benefits of rt-PA are that it shows a critical role in inhibiting neuronal apoptosis and promotes functional recovery in later phase stroke [46].

5.2. Mechanical Thrombectomy (MT).

Mechanical treatment, an endovascular procedure called endovascular thrombectomy (EVT), is another option to remove a clot in eligible patients with a large vessel occlusion. A review of randomized controlled trials concluded that EVT significantly improves the outcome for eligible patients [47]. The procedure involves the surgeon threading a catheter through a blocked artery in the brain. The stent will open and grab the clot, which will allow removal of the stent with the trapped clot [48]. This method can restore vascular patency (i.e., the degree to which blood vessels are not obstructed) of the vessels with a success rate between 41% and 54%. For patients with AIS caused by a proximal large artery occlusion in the anterior circulation who can be treated within 24 h of the time they were last known to be at their neurologic baseline, treatment with intra-arterial mechanical thrombectomy is recommended whether or not the patient received standard treatment with rt-PA. According to guidelines, eligible rt-PA patients should receive this first-line therapy without delay even if mechanical thrombectomy is being considered.

5.3. Treatments for hemorrhagic stroke.

Treatment of hemorrhagic stroke involves controlling bleeding in the brain (i.e., lowering blood pressure) and reducing the pressure caused by the bleeding. Surgical procedures of placing clamps at the base of the aneurysm are performed or less invasive methods, such as “coil embolization” are performed where a catheter is threaded through an artery in an arm or leg and guided into the brain [49]. When the catheter is guided to the place of bleeding, it will deposit a mechanical agent, such as a coil to prevent rupture of the vessel.

6.0. Blood Brain Barrier (BBB) and stroke

It is important to properly understand the role of the BBB to recognize the effectiveness of potential diagnostic markers for brain diseases, such as stroke. The BBB’s primary goal is to create a restrictive barrier between the central nervous system (CNS) and the rest of the body to prevent the entry of undesirable blood-borne factors while transporting toxic products back into circulation. The BBB’s microvessels are made of endothelial cells that are linked by tight junctions (TJ). The neighboring glial cells including astrocytes and microglial are also important to BBB function. All of these components together are known as the neurovascular unit. The cellular composition of the BBB is illustrated in Figure 4A [50].

Figure 4.

(A) Cellular constituents of the BBB. Cerebral endothelial cells form tight junctions that restrict the paracellular pathway. Pericytes are distributed along the length of the cerebral capillaries and partially surround the endothelium. Both the endothelial cells and the pericytes are surrounded by a basal lamina. Astroglial end feet form a complex network surrounding the capillaries and provide the cellular link to the neurons. Microglia are immune cells in the CNS (reprinted from [50], Copyright 2017 with permission from Elsevier).(B) Timing of events after a stroke event [51].(C) Conceptual model of the relationship of biomarkers, surrogate endpoints, and the process of evaluating therapeutic interventions (reprinted from [52], Copyright 2001 Wiley). (D) Properties of biological markers [52].

The BBB plays an important role in the immune system of the brain. The TJ between the endothelial cells that restrict blood-borne substances from entering the brain can be affected by brain injuries including stroke. Under stroke conditions, the BBB TJ integrity decreases, which results in increased paracellular permeability. This will cause ionic dysregulation, inflammation, oxidative and nitrosative stress, enzymatic activity, and angiogenesis [53]. Breakdown of the BBB also allows for passage of biomarkers from the neurons and the glial cells into circulating blood [54]. Evidence suggests leukocytes can move through the compromised BBB in humans in 48 – 72 h following a stroke event and it is hypothesized that the accumulation of these leukocytes is a reason for tissue damage and prevention of the blood flow after restoration [55]. Moreover, biomarkers like matrix metalloproteinase-9 (MMP-9) play a biphasic role in stroke by disrupting the BBB during the initial phases of the stroke event and promotes vascular growth during recovery phases. Timing of events that take place following an AIS is listed in Figure 4B [51].

7.0. Molecular and cellular biomarkers of stroke

A molecular and cellular biomarker is defined as a broad category of medical signs with objective indications of medical state displayed by the patient that can be measured accurately and reproducibly [56]. Examples of molecular and cellular biomarkers are genes, blood cells, enzymes, proteins, peptides, or hormones. There can be upregulation or downregulation of these biomarkers resulting from a diseased state.

As discussed in previous sections, current diagnostic techniques for stroke involve neuroimaging techniques with some displaying poor clinical sensitivity and specificity, the long time required to carry out the imaging, highly subjective interpretation of images, and the need for highly trained personnel to carry out the measurement. Thus, it is important to find alternatives to neuroimaging to allow for making diagnostic decisions within the therapeutic time window mandated by most stroke therapeutic strategies.

Liquid biopsies (LB) are generating a significant amount of interest in the medical community due to the minimally invasive nature of acquiring biomarkers and the fact that they can enable precision decisions on managing a variety of diseases, including the oncology and non-oncology-related diseases, such as stroke [57,58]. LB of peripheral-blood-based biomarkers allows for easy accessibility of a particular biomarker. The LB biomarker would ideally appear in blood early following the stroke event and allow for high clinical sensitivity and specificity for diagnosing both types of stroke.

Discovery of new biomarkers and a diagnostic test developed for these biomarkers is challenging for several reasons, including the complexity of stroke conditions, the short time allowed to make a clinical decision, and the function of the BBB, which may delay the appearance of some markers into peripheral blood. Hence, the development of a rapid and simple diagnostic test will be extremely useful for pre-hospital or emergency site screening before treatment resulting in a higher percentage of eligible patients receiving rt-PA therapy.

Many aspects should be considered when selecting a biomarker for stroke. The major one is the time of appearance of the marker in peripheral blood following the stroke event and the ability of it to move through the BBB, if required. Unfortunately, there is no FDA approved molecular diagnostic test currently available for stroke.

In clinical studies, the sensitivity and specificity are different from the analytical sensitivity and specificity. The clinical sensitivity provides information on the positivity of the clinical test, whereas the clinical specificity provides information about the propensity to detect false positives [59]. Functional relationships for calculating these two clinical figures-of-merit are shown below.

$$Clinicalsensitivity=\frac{Truepositives}{Truepositives+Falsenegatives}$$

$$Clinicalspecificity=\frac{TrueNegatives}{Truenegatives+FalseNegatives}$$

An ideal biomarker or a panel of biomarkers appearing quickly in the blood stream in detectable quantities using the appropriate analytical technique should answer the following questions [60]: (1) Does the patient have a stroke? For example, the test must be able to differentiate stroke from SMs. (2) What type of a stroke is it? Is it AIS or hemorrhagic stroke? (3) Is there a need for thrombolytic treatment and is there a risk of recurrence? A conceptual design illustrating the relation of a biomarker with a clinical end point is shown in Figure 4C.

Based on this concept, a selected biomarker and test using it should have properties that are summarized in Figure 4D with five key characteristics: (i) Adds independent clinical information; (ii) accounts for a large proportion of the risk associated with a given disease or condition; (iii) be reproducible; 4) be sensitive; and 5) be readily available [52].

Biomarkers for stroke can be categorized into three groups: (i) Markers that measure the changes in the nervous system via brain imaging; (ii) molecular/cellular biomarkers; and (iii) pharmacodynamic biomarkers (biomarkers indicative of certain pharmacological responses useful in drug development) [61].

At present, no single biomarker with discriminative characteristics is robust enough to be clinically used in the diagnosis and management of patients with stroke. Sub-groups of these markers are shown in Figure 5A. Several biomarkers are released into the CSF and blood stream as a result of different brain diseases, hence, they may not be specific to stroke only. However, biomarker candidates for stroke may be rationally identified to reflect components of the ischemic cascade [62]. In the next sections, we will discuss the molecular biomarkers found in body fluids such as blood, plasma, serum, and CSF for stroke diagnosis.

Figure 5.

(A) Potential biomarkers of stroke are categorized by their role in the ischemic cascade. Representative markers for each category (neuronal injury, glial activation, lipid peroxidation, inflammatory, and hemostasis/endothelial dysfunction) are illustrated. IL 5 interleukin; NMDA 5 N-methyl-D-aspartate; PARK 5 Parkinson disease protein; TNF 5 tumor necrosis factor (Reprinted from [62] Copyright 2012 Wiley). (B) Release of NSE in acute stroke. Data shown as means ±95% CI. Shaded areas indicate the respective reference range, 12.5 μg/L (reprinted from [69], Copyright 2004 with permission from Elsevier).

7.1. Markers of glial activation, inflammation and oxidative stress.

After a stroke event, proteins are released from neurons and glia, which will enter into the blood stream but only after sufficient damage to the BBB occurs to allow them to pass through it [63]. Quantitation of these proteins in blood can aid in diagnosing stroke. Glial activation, neuronal injury, oxidative stress, and release of inflammatory mediators are some of the early events that take place after an AIS event [63]. S100B, glial fibrillary acidic protein, and myelin basic protein are considered to be markers that are relatively specific to glial function and studies have shown that these markers can be used for predicting risk of hemorrhagic transformation, prognosis, infarct volume, and stroke diagnosis [64]. C-reactive protein (CRP), MMP-9, interleukin 6 (IL6), adhesion molecules, and tumor necrosis factor alpha (TNF-alpha) are nonspecific inflammatory markers released due to the neuro-inflammatory cascade that have been studied for prognosis and diagnosis of AIS [65]. Challenges with these markers are their latency in appearance in circulation due to the need for sufficient damage required to the BBB to allow them to pass through it. For example, S100B latency in release and lack of specificity to stroke conditions limits its use in the clinic [66].

Oxidative stress and lipid peroxidation take place because of neuro-inflammation during neuronal injury. Some of the biomarkers related to these events are redox sensitive molecular chaperones, lipid oxidation products like malondialdehyde and oxidized low-density lipoproteins [67]. Unfortunately, many of these oxidative stress markers are not specific to brain injury. Attention is paid to lipids, however, they are highly enriched in the brain. As an example, the F₄-neuroprostane, which is a byproduct of free radical-induced oxidation of docosahexaenoic acid, is a fatty acid that is highly enriched in the CNS [68].

7.2. Markers of neuronal injury.

Following the initial events of glial activation and inflammation that occur as a result of a stroke event, pathological processes begin by which nerve cells are damaged or killed by excessive stimulation by neurotransmitters, such as glutamate and oxidative stress [62]. One of the extensively studied markers of neuronal injury is neuron specific enolase (NSE). Figure 5B shows the release pattern of NSE detected in AIS patients’ serum. As can be seen, the concentration of NSE did not reach the reference range until ~72 h, which is out of the therapeutic time window for rt-PA treatment [70]. Other neuron specific markers are N-methyl-D-aspartate receptor (NMDA-R), Visinin-like protein I, and heart fatty acid binding protein [71]. Although these biomarkers show high specificity to stroke conditions, the delayed time course of neuronal injury and their appearance in peripheral blood make these markers not useful for early diagnosis of stroke (see Table 4).

Table 4.

Biomarkers for diagnostic and prognostic evaluation of AIS patients.

Biomarker	Biomarker Function	Clinical Information	Bio-fluid Tested/Method	Ref.
Markers of Glial Activation
S100B	Calcium binding protein expressed in astrocytes and oligodendrocytes	Level decreased from day 3 following AIS (p < 0.0001). The significant differences in S100B level between days 3 and 5 from AIS vs. day 10 (p < 0.001). AIS n= 56, control n=38	Serum/ELISA	[81]
Glial fibrillary acidic protein (GFAP)	Expressed by astrocytes	Levels significantly higher in HS patients (P=0.0057), AIS n= 83, HS n=14	Plasma/ELISA	[82]
Myelin basic protein (MBP)	Myelin sheath protein	Patients with AIS had significantly higher levels of MBP vs. Controls (P value < 0.001), AIS n=20, control n=20	CSF/ ELISA	[83]
Markers of Inflammation
C-reactive protein (CRP)	Acute phase protein	Level in AIS patients statistically higher than in HS patient group (P<0.0001). AIS n= 32, HS n=32	Serum/Immuno-nephelo-metry	[84]
IL-6	Inflammatory cytokine	A positive correlation between IL-6, NIHSS and modified Rankin Scale of the AIS patients (P < 0.001, r = 0.6). Correlation between IL-6 level and infarction size in brain as evaluated by MRI (P < 0.001, r = 0.7). AIS n=45	Serum/ELISA	[85]
TNF-alpha	Inflammatory cytokine	Significant different level in AIS vs. control. AUC: 0.99, P < 0.0001. Significantly elevated level in HS vs. control. AUC: 1.0, P < 0.0001. AIS n= 262, HS n=42, Control n=200	Plasma/ELISA	[86]
ICAM-1	Immunoglobulin super family members	Significantly elevated in AIS vs. healthy controls. P <0.05, AIS n=262, HS n= 42, Control n=200	Plasma/ELISA	[86]
VCAM-1	inflammation of adhesion molecules	Sensitivity and Specificity of 90% for discriminating AIS from non-stroke patients. AIS n=65, control n=157.	Plasma/ELISA	[87]
Matrix metalloproteinase-9 (MMP-9)	Biomarker of the severity of acute brain ischemia.	Elevated mRNA expression level; correlated with the size of brain infarct lesion, poor neurological outcome and hemorrhagic transformation after thrombolytic therapy. n=21 Elevated mRNA level; higher in HS patients than in AIS. Stroke n=126, Control n=9	Blood PBMC/qPCR	[88,89]
Markers of oxidative stress
PARK-7	Redox-sensitive molecular chaperone	Higher diagnostic potential with an AUC of 0.897, p < 0.001. AIS n=72 Control n= 78	Plasma/ELISA	[90]
Malondi-aldehyde (MDA)	Lipid peroxidation product	Non-survivors (n = 29) showed higher serum higher serum MDA levels (p = 0.004) than survivors (n = 29).	Serum	[91]
Oxidized low density lipoprotein	Lipid peroxidation product	Median values significantly higher in patients with ICH than in patients with AIS (P < .0001). ICH n=7, AIS n=9	Serum/ELISA	[92]
Markers of neuronal injury
Neuron specific enolase (NSE)	Neuronal glycolytic enzyme	Significantly elevated in AIS patients vs. healthy control p≤0.05, AIS n=100, control n=101	Serum/ELISA	[93]
Heart fatty acid binding protein (H-FABP)	Involved in intracellular fatty acid transport	Elevated in stroke group vs. control: P < 0.001. Levels correlated with initial NIHSS score (r=0.46, P<0.01). AIS n=111, Control n=127	Plasma/ELISA	[94]
NMDA receptor	Cytosolic protein, Excitotoxic receptor	Sensitivity of 92% specificity of 96%, AIS n=192, Control n=100	Plasma/ELISA	[95]
Markers of Hemostasis and Endothelial dysfunction
Thrombomodulin (sTM)	Endothelial cell glycoprotein (anticoagulation properties)	Plasma levels significantly higher in AIS patients than controls (P < 0.005). AIS n=93, Control n=76	Plasma/ELISA	[96]
D-dimer	Fibrin degradation product	Medians higher in patients with cardioembolic stroke than in those with other etiologies (p<0.001), AIS n=98 Control n= 0	Plasma/ELISA	[97]
Von Willebrand factor (VWF)	Glycoprotein involved in platelet adhesion stabilization	Levels significantly elevated in severe AIS (based on NIHSS) P = 0.013, AIS n=131, Control n= 0	Plasma/Immuno-turbidimetric	[98]
Miscellaneous markers
Natriuretic peptides (ANP, BNP)	Vasoactive peptide hormones	A statistically significant difference between stroke patients group and control (p < 0.001), AIS n=40, Control n= 30	Plasma/Immuno-assay	[99]
N-terminal brain natriuretic peptide (NT-proBNP)	Associated with underlying cause of brain ischemia	Independently associated with the diagnosis of AIS stroke when compared to patients with HS (P<0.001). AIS n=767, stroke mimics n=115, HS n=100, control n=23	Plasma/ Ab-based (Search-Light)	[100]
Lipoprotein associated phospholipase A2 (LpPLA2-M)	Hydrolytic enzyme	LpPLA2-M levels higher compared to controls, P=0.04). Sample n=167	Blood/ELISA	[101]
Calcium	Physiological ion, i.e., messenger that regulates many processes	The highest delayed Ca2+ quartile (versus lowest) was associated with lesser stroke severity and better 3-month functional and independence scale outcomes (all P<0.001). No significant outcome differences noted among early Ca2+ levels. AIS n=659, control n= 167	Serum	[102]
Free hemoglobin (Hb)	Erythrocyte protein	Hb alpha-chain and beta-chain differentially expressed between stroke patients and controls. No significant correlation (p > 0.05) between Hb chains and the NIHSS, TOAST, mRS, stroke risk factors, infarct volume, infarct location and laboratory data. Sensitivity 70.2%, Specificity 85.3%, AIS n=47 Control n= 34	Serum/MALDI-TOF, MS	[78]
Asymmetric dimethylarginine (ADMA)	Endogenous inhibitor of nitric oxide synthase	ADMA higher in stroke patients than in controls, elevated levels at day 3 and 7 indicative of an unfavorable clinical outcome. Specificity 75%, sensitivity 60% 3 days post stroke event. AIS n=67, control n=32	Plasma/HPLC-MS-MS	[103]
Parkinsonism Associated Deglycase (PARK7) and nucleoside diphosphate kinase A (NDKA)	PARK7: RNA binding protein regulatory subunit NDKA: Elevated in neurodegenerative disease.	Increase in concentration 3 h of stroke onset. An increase in concentration of both markers observed in each type of stroke, HS, TIA and AIS, compared with controls (P <0.001). PARK7: Sensitivities of 54%-91% and specificities of 80%-97%.NDKA: Sensitivities of 70%-90% and specificities of 90%-97%. AIS n=234, TIA n=153, HS n=235, control n=165	Plasma/ELISA	[67]
Panel of markers: insulin-like growth factor-binding protein-3 (IGFBP-3), tumor necrosis factor receptor-1 (TNF-R1), Fas ligand (FasL), S100B, Heat shock 70 kDa protein-8 (Hsc70), apolipoprotein CIII, and neuron cell adhesion molecule (NCAM)		Real stroke vs. stroke mimics: AUC= 0.742, AIS vs HS AUC=0.757. AIS n=767, stroke mimics n=115, HS n=100, control n=23	Plasma/ Ab-based (Search-Light)	[100]
Ubiquitin C-terminal hydrolase (UCH-L1) and glial fibrillary astrocytic protein (GFAP)	GFAP: brain-specific astrocytic intermediate filament protein, UCH-L1 is a cytoplasmic deubiquitinating enzyme of neurons	UCH-L1 and GFAP levels elevated in ICH patients vs. controls (P < 0.0001). GFAP differed in ICH vs. AIS (P < 0.0001), AUC = 0.86 within 4.5hrs of symptom onset, Sensitivity = 61%, Specificity = 96%. Higher GFAP levels associated with stroke severity and history of prior stroke. ICH n=45, AIS n=79, SAH n=5, TIA n= 3, control n=57.	Serum/ELISA	[104]
Copeptin	neuroendocrine marker	High level predicted unfavorable outcome P < 0.001, AUC 0.83 Concentrations along with the NIHSS better predictor of functional outcome and mortality within 90 days than the clinical scale or the biomarker alone. AIS n=783, Control n=359	Plasma/ELISA	[105]
circulating cfDNA	Marker of cell death via apoptosis or necrosis.	Levels correlated with severity of stroke at the time of admission (P=0.032) and poor outcome (P=0.001). AIS n=26, control n=0 cfDNA marker added to the clinical predictive model improved AIS discrimination (p = 0.009). AIS n=54, control n=15	Plasma/qPCR	[106] [107] [108]

Notes: AIS – acute ischemic stroke, HS – hemorrhagic stroke, ICH - Intracerebral hemorrhage, MALDI - Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, HPLC-MS/MS- high-performance liquid chromatography-tandem mass spectrometry, CNS - central nervous system, SAH- subarachnoid hemorrhage, TIA- transient ischemic attack, ELISA- enzyme-linked immunosorbent assay, qPCR- quantitative polymerase chain reaction

7.3. Markers of hemostasis and endothelial dysfunction.

Under normal physiological conditions, platelets do not interact with endothelial cells and this provides a natural resistance to thrombosis. During cerebral ischemia, hypoxic endothelial cells upregulate cell adhesion molecules and matrix proteins, and platelet-vessel wall interactions develop. A key step in platelet adhesion following vascular injury is binding via surface receptors to endothelial von Willebrand factor (vWF) and adhesion of platelets to collagen, which can result in reduction of blood flow and delayed injury. Some of the classic hemostatic markers that are identified as potential biomarkers of stroke are thrombomodulin, D-dimer, fibrinogen, fibronectin, and vWF [72]. A few studies have been conducted with plasminogen activator inhibitor-I (PAI-I), D-dimer, and thrombin-activatable fibrinolysis inhibitor (TAFI), where researchers identified patients having risk of hemorrhagic transformation after rt-PA administration [73]. Asymmetrical dimethylarginine (ADMA), which is also a marker of dysfunction, has been studied as a potential marker for subclinical cerebrovascular injury [74]. However, ADMA levels are also increased in people with hypercholesterolemia, atherosclerosis, hypertension, chronic heart failure, diabetes mellitus, and chronic renal failure (Table 4) [75].

7.4. Miscellaneous markers.

There are other biomolecules that do not belong in the aforementioned categories and have been shown to be potential biomarkers of stroke. As an example, atrial natriuretic peptide (ANP), a cardiac hormone whose main function is to lower blood pressure and control electrolyte homeostasis was elevated following a stroke event [76]. Plasma concentrations of ANP were significantly higher in stroke patient samples compared to healthy controls. Another marker, brain natriuretic peptides (BNP), also was shown to be elevated after AIS and subarachnoid hemorrhage. BNP is also used as a marker for diagnosing congestive heart failure, therefore it is still unclear whether the increased levels of BNP are of cardiac or brain origin [76]. Lipoprotein-associated phospholipase A2 is an enzyme that hydrolyzes oxidized phospholipids. This enzyme has also been studied as a biomarker for ischemic stroke [77]. Free hemoglobin has also been suggested as a potential marker for stroke because it has shown increased levels in AIS patients compared to healthy controls [78]. Table 4 summarizes currently reported biomarkers of stroke and their function.

7.5. microRNA (miRNA) markers.

miRNA are recognized as important post-transcriptional regulators of gene expression. miRNA, through a variety of mechanisms like mRNA degradation and regulation of translation, controls the expression of ~30% of all transcripts. Following miRNA discovery in 1993, research has shown that miRNAs are one of the key players in cell differentiation and growth, mobility, and programmed cell death [79]. In a recent study, microarrays were utilized to study whole blood miRNA changes specific to AIS [80]. It was discovered that miR-122, miR-148a, let-7i, miR487b were detected at lower levels, while miR-363 and miR-487b were abundant in AIS patients.

8.0. Gene expression profiling in peripheral blood cells as biomarkers of stroke

Gene expression profiling measures the activity of genes in terms of their transcript numbers (i.e., mRNA) that are expressed in a cell at any given moment. Alterations in gene expression in terms of upregulation and/or downregulation of gene activity and monitoring the changes in mRNA expression levels can be studied in response to many diseases and this information can be used as biomarkers for disease management.

mRNA biomarkers offer numerous advantages over protein-based biomarkers. One such advantage is that mRNA levels can be easily monitored and detected even at low expression levels and mRNA expression analyses can offer high throughput because thousands of genes can be analyzed at the same time. Additionally, mRNA is transcribed more quickly in response to disease when compared to protein translation. Rapid multiplex RNA tests could make this a possibility for real-time testing. The precedence of rapid RNA-based testing exists as there are already diagnostic tests for HIV. In addition, a relatively new company, IschemiaCare (https://www.ischemiacare.com/), uses a CLIA-certified laboratory development test of mRNA expression from peripheral blood to aid in the diagnosis and treatment of stroke. In the next sections, we will review the work performed on using mRNA secured from biological cells found in peripheral blood as biomarkers for stroke.

8.1. Peripheral blood mononuclear cell (PBMC) mRNA markers.

Moore et al. [109] were the first to publish gene expression profiling in circulating PBMCs with regards to stroke. mRNA from PBMCs isolated from stroke patients were interrogated using microarrays. Results demonstrated that within 72 h after an AIS event, there was an up regulatory response of certain genes in PMBCs. Specifically, 22 genes were identified as potential biomarkers for stroke diagnosis with 78% clinical sensitivity and 80% clinical specificity [109].

In two other studies performed by Barr et al. and Tang et al., 9 genes and 17 genes, respectively, were upregulated during an ischemic stroke event [110,111]. Some common genes, such as NPL, ETS2, VCAN, MMP9, S100A12, ARG1, CA4 and LY96, were identified in these studies (Table 5).

Table 5.

Stroke related transcript panels identified in microarray gene expression studies.

Transcript (Moore et al. a)	Gene regulation	Transcript (Tang et al. b)	Gene regulation	Transcript (Barr et al c)	Gene regulation
CD163 PLBD1		Hox 1.11 CKAP4		CA4 CCR7	Up
CD163 PLBD1 ADM KIAA0146 APLP2 NPL FOS TLR2 NAIP CD36 DUSP1 ENTPD1 VCAN CYBB IL13RA1 LTA4H ETS2	Up	Hox 1.11 CKAP4 S100A9 MMP9 S100P F5–1 FPR1 S100A12 RNASE2 CA4 LY96 SLC16A6 HIST2H2AA3 ETS2 BCL6 PYGL NPL	Up	ARG1	Down
CD14–1 CD14–2 BST1 CD93 PILRA FCGR1A		ARG1	Down

Note:

^a-[109]

^b-[110]

^c-[111]

In terms of the timeframe of gene activity dysregulation resulting from stroke, work conducted by Moore et al. showed over 85% accuracy for identifying stroke patients as early as 1–3 h after stroke symptoms when tested on a group of AIS patients. A study performed by Tang et al. showed changes in 17 genes in 1–3 h of symptoms that persisted at 5 – 24 h. In another study, a panel of 30 genes were identified for intracerebral hemorrhage (ICH) using reverse transcription quantitative PCR (RT-qPCR) and the investigators were able to obtain >85% accuracy for ICH diagnosis [112]. Furthermore, the authors identified two genes, Amphiphysin (AMPH) and Interleukin 1 Receptor Type 2 (IL1R2), that were expressed differently in hemorrhagic and ischemic stroke patients [112].

8.2. mRNA markers in subpopulations of circulating leukocytes.

In a recent study by Adamski et al. [113], high throughput RT-qPCR (HT RT-qPCR) was used to verify the results of microarray studies supporting the use of mRNA biomarkers for early stroke diagnosis. The authors used subpopulations of circulating leukocytes and interrogated their gene expression levels. The hypothesis behind the study was that selecting certain leukocyte subsets would provide more specific expression differences for stroke diagnosis when compared to PBMCs. As the innate immune response is known to be the major system involved in AIS, it was hypothesized that cells of the innate immune response may be the main leukocyte subsets showing altered gene expression responding to stroke [10].

The investigators used density gradient centrifugation to isolate PMBCs and granulocytes from whole blood. Then, the granulocyte fraction was purified to contain only CD15+ cells and the PMBCs were used to isolate fractions of CD14+, CD4+, CD20+ and CD8+ leukocyte subpopulations. Total RNA (TRNA) was extracted from these cells and 40 transcripts identified in earlier studies were selected for the analysis (Table 5).

Using HT-RT qPCR results, certain genes were significantly upregulated in AIS patients in 4 leukocyte subpopulations. In CD15+ granulocytes, 14 genes were upregulated while in CD8+ T-lymphocytes 16 genes were upregulated. Figure 6A illustrates gene expression alterations in AIS patients. Furthermore, in hierarchical cluster analysis, 43 clusters of transcripts were identified specific to cell subsets that showed a significant discrimination between AIS and healthy controls Figure 6A (b–d) shows validation results performed using these genes demonstrating the overall accuracy of the 3-gene cluster classified stroke with a clinical sensitivity of 89% and a clinical specificity of 67%. Changes in expression of certain genes indicative of stroke were observed in AIS patients and provided 66%, 87%, and 100% clinical sensitivity for 2.4 h, 5 h and, 24 h following stroke onset using CD8+ T-cells [110]. Based on these findings, upregulation of certain gene subsets provides high sensitivity for stroke diagnosis and serves as a promising method for stroke early diagnosis. Moreover, gene expression analysis has great potential to be applied to point-of-care testing (POCT) owing to the accessibility of blood samples, high accuracy for AIS detection, and quantitative gene expression changes.

Figure 6.

(A) (a) Hierarchical cluster analysis and heatmap of fold changes in expression of 41 genes, in 6 leukocyte subsets, between IS (n=18) and control subjects (n=15). (Reproduced from reference 148). (b) Boxplots demonstrating threshold values for defining elevated expression of each of the transcripts (CA4, NAIP, MMP-9). (c) Bar graphs depicting the number of transcripts elevated in stroke patients and healthy control subjects. (d) ROC analysis for Cluster 1 for stroke classification revealed that the AUC was 0.813. Elevation of 3 or more transcripts gave the greatest sensitivity and specificity (Reprinted from [113] Copyright 2017 The Medical Research Archives). (B) SEMs of a cell selection chip containing high-aspect ratio channels with a sinusoidal architecture. (Reprinted from [114] Copyright 2015 American Chemical Society). (C) Parallel arrangement of cell selection microchips for the isolation of T cells (using anti-CD4 antibodies) and neutrophils (using anti-CD66b antibodies). (Reprinted from [114] Copyright 2015 American Chemical Society).(D) Gene expression profiling of selected genes from T cells and neutrophils. The mRNA transcripts were harvested from selected cells and subjected to RT-qPCR. (Reprinted from [114] Copyright 2015 American Chemical Society).

8.3. Microfluidics for cell selection with potential for clinical stroke diagnostics using mRNA.

In clinical applications, it is important to employ portable devices that can also provide full assay automation even at the point-of-care without sacrificing the assay’s analytical and clinical figures-of-merit. Pullagurla et al. [114] demonstrated a microfluidic device for the selection of leukocyte subsets directly from peripheral blood and gene expression analysis to distinguish AIS from hemorrhagic stroke. The device for cell selection used antibodies covalently attached to the surface of the microfluidic and enabled the affinity selection of leukocyte subsets with high purity. The device consisted of a series of sinusoidal channels and was designed to simultaneously isolate multiple cell types from blood. In this study, CD4+ T cells and CD66b+ neutrophils from whole blood were isolated as shown in Figure 6B–D. Gene expression of mRNA isolated from both subpopulations demonstrated that CD4+ T-cells and CD66b+ neutrophils differentially expressed transcripts that hold promise for diagnosing and differentiating between ischemic and hemorrhagic strokes [115].

As discussed in previous sections, time is one of the most critical factors in stroke diagnosis. While microarrays can provide high throughput screening of thousands of genes, it required ~11 h to perform the analysis. Also, special instruments are needed and experienced personnel to perform the analysis, which is not conducive to POCT [116]. Microfluidic platforms can be developed for POCT because they deliver faster analysis times and ease of use with minimum equipment requirements.

9. Conclusion

Among the two types of stroke, hemorrhagic and AIS, ~85% patients experience AIS for which rapid diagnosis is essential. The current standard-of-care for stroke patients is to undergo CT to rule out hemorrhagic stroke. CT clearly shows hyper-dense lesions due to hemorrhagic stroke but is much less sensitive for AIS, and other medical conditions such SM and old infarctions. As such, CT has only 26% clinical sensitivity for AIS [117]. MRI provides higher sensitivity (83%) for both AIS and hemorrhagic stroke, but is more time consuming than CT and is not widely used in an emergency setting.

Clot-busting thrombolytic treatment using rt-PA is the cornerstone of AIS therapy, but is contraindicated for hemorrhagic stroke [118]. Unfortunately, rt-PA, approved by the USA FDA in 1996 reaches only ~7% of AIS patients because the timeframe for treatment is only 4.5 h after the onset of stroke symptoms and current diagnostics for AIS do not meet that time constraint [119].

In the event of brain disease or injury, the immune system triggers production and release of proteins into the blood. Several different types of molecular biomarkers are dysregulated following a stroke event, such as proteins/peptides responding to neuronal damage. Proteins involved in AIS pathogenesis include markers related to glial activation, inflammation, oxidative stress, neuronal injury, and endothelial dysfunction. Unfortunately owing to biology, these proteins become elevated in plasma 6–12 h from symptom onset with continuous increase after a few days. This delay in response to a stroke event in blood proteins challenges their use as biomarkers for early detection of AIS. Additionally, some of these protein markers are not specific exclusively to AIS.

An interesting approach for stroke diagnostics uses leukocytes and their genetic transcripts. In the event of AIS, leukocytes are recruited to the injury site and become activated [120]. Evaluation of different leukocyte subpopulations in stroke patients and mouse models provided important insights into mRNA expression changes in response to stroke [109]. Based on these studies, gene panels were discovered specific to stroke (~85% accuracy for stroke diagnosis). Critically, changes in mRNA expression were observed ~3 h following stroke onset, thus preceding the release of proteins into blood.

10. Expert Opinion

In vitro diagnostic tests for the early detection of AIS would improve outcomes for these patients. For a diagnostic to be a viable alternative to neuroimaging methods, it must be ideally performed at the point-of-care and require results to be delivered shortly upon identification of the suspected stroke symptoms. LBs are generating a significant amount of interest in the medical community due to the minimally invasive nature of acquiring blood borne biomarkers, potential for frequent testing, and the fact that they can enable precision decisions on managing a variety of diseases, including AIS [121]. A rapid, sensitive, and specific test that could deliver the diagnosis within minutes from the onset of stroke would be very valuable for deciding which patients are appropriate for rt-PA therapy and/or endovascular thrombectomy.

New LB markers, such as extracellular vesicles (EVs) and their miRNA or mRNA cargo could potentially provide a more timely and detectable appearance of stroke-related markers fore diagnostics. EVs released by cells through an endosomal pathway or by budding from the plasma membrane carry molecular cargo characteristic of the cell from which it originated. Each EV subtype is classified based on their biogenesis and release pathway and can carry different types of miRNAs and mRNAs [122]. EVs are LB markers found in different body fluids, including blood [123]. In the CNS, EVs maintain normal neuronal function and are involved in neurodegenerative diseases. For example, EVs released from CD8+ T-cells play key roles in CNS homeostasis, stroke pathology, and subsequent stroke recovery [124]. Therefore, EVs isolated from blood plasma could provide real time information on changes occurring in the brain [125].

Following a stroke event, EVs that originate from activated leukocytes responding to tissue damage inside the vasculature of the brain could be a viable source of miRNAs and/or mRNAs for stroke diagnosis. EVs could potentially be a more abundant source of mRNA transcripts due to their high abundance in blood plasma, which can be used for expression profiling at an earlier phase following stroke compared to cell-of-origin.

Future work evaluating the time-evolution of mRNAs or miRNAs isolated from EVs would allow identification of gene panels specific for particular types of stroke. Analysis of the mRNA cargo from EVs would differentiate between the major stroke subtypes, AIS and hemorrhagic. Also, the test using EVs should rule out SMs and potentially identify TIA. Owing to the importance of rapid treatment, future tests based on EVs’ mRNA cargo would have to adhere to national guidelines that recommend diagnosis of stroke patients and therapy.

Article highlights:

• Eighty five% of stroke patients experience acute ischemic stroke (AIS) for which rapid diagnosis is essential to allow for thrombolytic treatment using rt-PA.

• rt-PA is contraindicated in hemorrhagic stroke patients, therefore differentiation between AIS and hemorrhagic stroke is essential.

• Short timeframe (4.5 h) for rt-PA treatment is a major contributing factor leading to the relatively small number of patients (~7%) receiving this treatment in time.

• Delay in diagnosis are due to deferral in seeking medical help, lack of specialized imaging equipment in some medical venues, or lack of around the clock medical personnel to perform the test.

• Computed tomography (CT) can rule out hemorrhagic stroke but is much less sensitive for AIS.

• Magnetic resonance imaging (MRI) provides 83% sensitivity to AIS but it is not widely used in an emergency setting.

• There is no FDA approved molecular diagnostic test for stroke diagnosis.

• Protein markers associated with AIS related to glial activation, inflammation, oxidative stress, neuronal injury, and endothelial dysfunction become elevated in blood 6–12 h after symptom onset, therefore, are not adequate to accommodate the short diagnostic timeframe for AIS.

• Leukocytes recruited to the brain injury site show gene expression differences characteristic of either AIS or hemorrhagic stroke.

• Evaluation of different leukocyte subpopulations in stroke patients and mouse models identified gene panels specific to stroke (~85% accuracy for stroke diagnosis).

• Another liquid biopsy marker, namely extracellular vesicles (EVs), can provide information on mRNA or miRNA abundance distinct to different types of stroke.