Recent stroke biomarkers

1. Recent stroke biomarkers
By
Prof. Moustafa Rizk
Prof. of Clinical Pathology
Faculty of Medicine, University of Alexandria
10/19/2017 5:22 AM 1

2. Stroke is defined as
A clinical syndrome consisting of :
 Rapidly developing clinical signs of focal disturbance of
cerebral function
 Lasting more than 24 hours
 Or leading to death
 With no apparent cause other than that of vascular origin
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3. Stroke Types
 80% ischemic
 Thrombosis
 Embolism
 Hypoperfusion
 20% hemorrhagic
 Intracerebral
 Subarachnoid
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4. Ischemic Strokes
• Thrombosis-most common cause
• Etiology
– Atherosclerotic disease-most common
– Vasculitis
– Dissection
– Polycythemia
– Hypercoagulable states
– Infectious Diseases-HIV, TB, syphilis
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5. Ischemic Strokes
• 1/5th due to Embolism
• Etiology
–Cardiac
• Valvular Vegetations
• Mural thrombi- caused by
A-fib, MI, or dysrhythmias
• Paradoxical emboli
• Cardiac tumors-myxoma
–Fat emboli
–Particulate emboli – IV drug injections
–Septic Emboli
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6. Ischemic Strokes
• Hypoperfusion- less common mechanism
• Typically caused by cardiac failure
• More diffuse injury pattern
vs. thrombosis or embolism
• Usually occur in watershed
regions of brain
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7. Hemorrhagic Strokes
 Intracerebral hemorrhage (ICH)
- approx. 10% of all strokes
– Risk Factors
• HTN
• Increasing Age
• Race: Asians and Blacks
• Amyloidosis- esp. in the elderly
• AVMs or tumors
• Anticoagulants/Thrombolitic use
• History of previous stroke
• Tobacco, ETOH, and cocaine use10/19/2017 5:22 AM 7

8.  Subarachnoid hemorrhage (SAH)
–Result from rupture of
berry aneurysm or
rupture of AVMs
Hemorrhagic Stroke
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9. A transient ischaemic attack (TIA)
Is defined as ‘stroke symptoms and signs that resolve
within 24 hours’
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10. 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.
A biomarker has good clinical acceptance if the biomarker is :
Accurate
Acceptable to the patient
Easy to interpret by clinicians
Has a high sensitivity and specificity for the outcome it is
expected
Understanding of the differences in pharmacological
responses
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11. Types of stroke biomarkers
1- Biomarkers of oxidative damage
Ex. Oxidative stress index
F2-isoprostanes , Uric acid
2- Biomarkers of inflammation
Ex. Interleukin-6 , CRP
Toll-like receptors
3- Biomarkers of brain injury
Ex. Plasma microRNAs , S100β
Neuron-specific enolase
4- Lipids related Biomarkers
Ex. Apolipoprotein A-1
Fatty acid binding protein 4
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12. 5- Biomarkers of thrombus formation
Ex. D-dimers
Platelet reactivity
6- Biomarkers of cardiac function
N-terminal pro-Brain Natriuretic Peptide
High sensitivity troponin T
7- Biomarkers of stroke risk
Lipoproteinassociated phospholipase A2 (Lp-PLA2)
High levels of oxidized low-density lipoprotein (oxLDL)
Peptide midregional proadrenomedullin (MR-proADM)
Asymmetric dimethylarginine (ADMA)
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13. 1-Biomarkers of oxidative damage
Oxidative stress index (OSI)
 The ratio of total oxidant status (TOS ) level to total
antioxidant status (TAS) level was considered as OSI.
 OSI was calculated according to the following formula:
OSI = TOS (μmol H2O2 Eq/L) / TAS (μmol trolox Eq/L)
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AIH/healthy adults 9.101 ± 5.829/2.096 ± 1.130 p=< 0.001
ACI/healthy adults 8.844 ± 6.585/2.096 ± 1.130 p=< 0.001
American Journal of Emergency Medicine 34 (2016) 2379–2383
Investigation of oxidant and antioxidant levels in patients with acute stroke in the emergency service

14. • F2-isoprostanes (A family of prostaglandin isomers)
Rise in F2-isoprostanes occurred as early as three
hours after ischemic stroke onset and remained
elevated for several days.
• Uric acid (an antioxidant role of urate)
Uric acid levels were inversely associated with the
extent of neurological deficits on admission and the
final infarct volume on CT/MRI scans
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15. 2- Biomarkers of inflammation
Interleukin-6
Released in the post-stroke setting and serves as a vital
messenger molecule between leucocytes, vascular
endothelium, and parenchyma resident cells.
Blood levels of IL-6 have been repeatedly associated
with poor outcome.
After stroke IL-6 concentration in blood has been
correlated with baseline stroke severity, highlighting
its plausible role as a biomarker of acute cerebral
injury.
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16. Toll-like receptors
• 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.
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A family of transmembrane pattern recognition receptors

17. 3- Biomarkers of brain injury
• Plasma microRNAs
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Differentially regulated miRNAs in ischemic stroke patients with different conditions i.e., acute
ischemic stroke cases and ischemic stroke cases in different time scales.
M. Vijayan, P.H. Reddy / Biochimica et Biophysica Acta 1862 (2016) 1984–1993
Microarray analysis and selective
TaqMan qPCR of miRNAs
Peripheral biomarkers of stroke: Focus on circulatory microRNAs

18. • Using microarray analysis , the researchers found that among the
836 miRNAs present on the array chip, 157 miRNAs were
differentially regulated in the stroke subjects.
• Among those miRNAs, 138 miRNAs were highly expressed, and 19
were poorly expressed. Of the highly expressed miRNAs, 17 were
upregulated and of the poorly expressed miRNAs, 8 were down
regulated.
• The researchers found that analysis of miRNA profiling revealed
the following key events that occur during stroke recovery:
regulation of hypoxia, angiogenesis, and erythropoiesis/
hematopoiesis.
• They concluded that these miRNAs could be used to differentiate
large artery, small artery, and cardio embolic strokes from each
other.
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M. Vijayan, P.H. Reddy / Biochimica et Biophysica Acta 1862 (2016) 1984–1993

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Total RNA was separately extracted from MRI(-) acute stroke patients, MRI(+) acute stroke
patients, and control subjects. Then, significant changes in the miRNA profiles were
analyzed using pairwise comparisons between the groups. Only the miRNAs showing 2-fold
changes or greater between all two groups were selected.
Journal of Stroke and Cerebrovascular Diseases, Vol. 23, No. 10 (November-December), 2014: pp 2607-2613
Circulating MicroRNAs as Novel Potential Biomarkers for Early Diagnosis of Acute Stroke in Humans

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Identification of miRNAs in plasma samples as potential biomarkers for the
diagnosis of acute stroke. The 17 candidate miRNAs screened by miRNA microarray
and qRT-PCR were separately detected by real-time RT-PCR in MRI(-) acute stroke
patients, MRI(+) acute stroke patients, and control subjects.
hsa-miR-106b-5P
hsa-miR-4306
hsa-miR-320d
hsa-miR-320e
Journal of Stroke and Cerebrovascular Diseases, Vol. 23, No. 10 (November-December), 2014: pp 2607-2613
23.90-fold

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Evaluation of plasma microRNAs hsa-miR-106b-5P for the diagnosis of acute stroke patients
A, ROC curves were drawn from the
plasma microRNAs from 60 MRI(-) acute
stroke patients
B, ROC curves were drawn from the
plasma microRNAs from 76 MRI(+) acute
stroke patients.

22. • S100β
• Largely expressed by astrocytes and, to a lesser extent,
by neurons, microglia, and oligodendrocytes.
• S100β is protective and trophic at low concentrations,
but can also be toxic and pro-apoptotic at high levels.
• The glial protein S100β was increased in ischemic
stroke patients who present 1-7 days after the insult.
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Serum Protein S100β is a Diagnostic Biomarker for Distinguishing Posterior Circulation Stroke from Vertigo of Nonvascular Causes
European Neurology 72(5-6):278-284 · October 2014

23. • Neuron-specific enolase
• Neuron-specific enolase (NSE) is a cytosolic brain enzyme
that functions as a glycolytic isoenzyme .
• NSE is confined solely in neurons under normal conditions
and only a negligible amount of NSE is physiologically
present in blood .
• 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.
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24. 4- Lipids related Biomarkers
• Apolipoprotein A-1
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Journal of Stroke and Cerebrovascular Diseases, Vol. 25, No. 6 (June), 2016: pp 1360–1365

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Median Apo A-I levels were lower in ischemic stroke cases versus controls (140 versus 175
mg/dL, difference of 35 mg/dL, 95% CI −54 to −16) and in ischemic stroke versus ICH
cases (140 versus 180 mg/dL, difference of 40 mg/dL, 95% CI −57 to −23)
Journal of Stroke and Cerebrovascular Diseases, Vol. 25, No. 6 (June), 2016: pp 1360–1365
Apolipoprotein A-I and Paraoxonase-1 Are Potential Blood Biomarkers for
Ischemic Stroke Diagnosis

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Median paraoxonase-1 was lower in ischemic stroke cases than in both ICH cases and
matched controls
Journal of Stroke and Cerebrovascular Diseases, Vol. 25, No. 6 (June), 2016: pp 1360–1365
Apolipoprotein A-I and Paraoxonase-1 Are Potential Blood Biomarkers for
Ischemic Stroke Diagnosis

27. Fatty acid binding protein 4
 Involved in coordination of lipid transportation and
atherogenesis.
 Expressed mainly at an inflammatory state in adipose tissue
 A certain amounts of FABP4 are also found in macrophages,
which are pathogenic constituents of atherosclerotic plaques.
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28. Fatty acid binding protein 4
 Previous studies have suggested that FABP4 was associated
with the following known cardiocerebrovascular diseases risk
factors: insulin resistance and obesity, hypertension,
atherosclerosis and diabetes.
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Circulating FABP4 levels are associated with stroke risk and clinical severity and
may represent an important pathophysiological mediator of atherosclerosis, which
may point to a new target of treatment options.
Although further studies are now needed to replicate these findings, data suggest
that FABP4 level shows potential as a novel biomarker for stroke risk and stroke
severity.
Journal of Neuroimmunology 311 (2017) 29–34
Fatty acid binding protein 4
Fatty acid binding protein 4 is associated with stroke risk and severity in patients with acute ischemic stroke

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Journal of Stroke and Cerebrovascular Diseases, Vol. 23, No. 5 (May-June), 2014: pp 910-918
Do we need a panel of biomarkers ?

31. Characteristics of the 5-variable model predicting ischemic stroke vs.
mimic, ischemic stroke + ICH vs. mimic
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Journal of Stroke and Cerebrovascular Diseases, Vol. 23, No. 5 (May-June), 2014: pp 910-918

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Receiver operating characterisitc curves. Discriminating capacity of 3 sets of
predictors including (1) red: age, race, sex; (2) green: the 5 tested biomarkers;
and (3) black: age, race, and sex plus the 5 tested biomarkers.
Journal of Stroke and Cerebrovascular Diseases, Vol. 23, No. 5 (May-June), 2014: pp 910-918
Ischemic stroke vs. Mimic Ischemic stroke+ICH vs. Mimic Ischemic stroke vs. ICH
A Blood-based Biomarker Panel to Detect Acute Stroke

33. THANK YOU
PROF./MOUSTAFA RIZK
10/19/2017 5:22 AM 33