stroke biomarkers that can help in early diagnosis

1. Stroke Biomarkers: a new era with
diagnostic promise?
By
Prof. Moustafa Rizk
Prof. of Clinical Pathology
Faculty of Medicine, University of Alexandria
24/3/2022
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2. How to Spot a Stroke
B = Balance: Does the person have a sudden loss of balance or
coordination?
E = Eyes: Do they have sudden double vision, or loss of vision in one or
both eyes?
F = Face: Is one side of the face drooping? Ask the person to smile.
A = Arms: Can they keep their arms up, or does one arm drift down?
S = Speech: Do they suddenly have difficulty speaking or is their
speech slurred?
T = Time to act!.
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Stroke Types

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Adnan I, et al. Acute Ischemic Stroke and COVID-19.Stroke. 2021;52:905–912.
Acute Ischemic Stroke and COVID-19
 A total of 8163 patients with confirmed COVID-19
among 27676 patients
 103 (1.3%) patients developed acute ischemic stroke
among 8163 patients with COVID 19.
 199 (1.0%) patients developed acute ischemic stroke
among 19513 patents in whom COVID-19 infection was
not diagnosed.
 Acute ischemic stroke was infrequent in patients with
COVID-19 and usually occurs in the presence of other
cardiovascular risk factors.

5. Interventions in ischemic stroke
 Thrombolysis with recombinant tissue
plasminogen activator (rt-PA)
 Aspirin given within 48 h
 Management of the patients within a dedicated
stroke unit
 Hemicraniectomy
 Recently endovascular clot retrieval
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6. 5:20 AM 6
Sources of the major candidate biomarkers.

7. 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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8. Types of stroke biomarkers
1- Biomarkers of brain injury
Ex. S100β , MMP-9 , Neuron-specific enolase , GFAP,
Plasma microRNAs
2- Biomarkers of inflammation
Ex. Interleukin-6 , Procalcitonin level , Tumor necrosis
factor-a (TNF-a)
3- Biomarkers of oxidative damage
Ex. Thioredoxin, F2-isoprostanes , Uric acid
4- Lipids related Biomarkers
Ex. Apolipoprotein A-1
Fatty acid binding protein 4
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9. 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
Lipoprotein a ssociated phospholipase A2 (Lp-PLA2)
High levels of oxidized low-density lipoprotein
Asymmetric dimethylarginine (ADMA)
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10. S100β
 The plasma S100β concentration in the ICH group was
significantly higher than in the IS group (p < 0.001).
 So S100β could serve as a potential biomarker for
differentiating between ICH and IS
 Plasma S100β concentration was significantly elevated
in patients with poor functional outcome vs. those
with favorable functional outcome (p < 0.001).
 So predicting short-term functional outcome after ICH.
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Zhou S, Bao J, Wang Y, Pan S. S100β as a biomarker for differential diagnosis of
intracerebral hemorrhage and ischemic stroke. Neurol Res. 2016 Apr;38(4):327-32.
1- Biomarkers of brain injury

11. MMP-9
• MMP-9 concentrations correlate positively to infarct size
and worse neurological outcome
• Serum MMP-9 concentrations ≥140 ng/ml were shown
to predict hemorrhagic transformation in rt-PA treated
ischemic stroke patients
• However, the rise of MMP-9 is not specific to ischemic
stroke, moreover its concentration is reported to peak at
24 h post stroke
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12. NSE
 Significantly raised in stroke patients compared to controls
and to correlate with infarct size and stroke symptom
severity
 Serum NSE levels assessed prospectively within 4.5 h of IS
symptom onset in rt-PA threated patients correlates with
NIHSS at 24 h , and lower serum NSE levels and NIHSS
scores were detected in patients with favorable
neurological outcomes
 Overall, NSE has a similar discriminatory profile to S100B
(high specificity and low sensitivity)
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13. GFAP (glial fibrillary acidic protein)
• The best candidate to date for differentiating
hemorrhage and ischemic stroke
• Using GFAP cut-off of 2.9 ng/l provided a
specificity of 96.3% and a sensitivity of 84.2% for
distinguishing ICH and IS .
• A meta-analysis including nearly 1,300 patients
confirmed the potential of measuring GFAP in the
blood in the early phase of stroke (samples drawn
<3 h from symptoms onset), to discriminate IS,
ICH.
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14. • Following an AIS, high levels of miRNA-124 were
attributed to different cellular processes, such as
inflammation, edema, cell death, and neurogenesis.
• Increased levels of miRNA-142-3p were correlated with
vascular hemorrhage, whereas low levels caused
abnormal vascular remodeling.
• Elevated levels of miRNA-126 were established as a
biomarker for AIS
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Plasma microRNAs

15. • 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 . Peripheral biomarkers of stroke: Focus on circulatory microRNAs .
Biochimica et Biophysica Acta 1862 (2016) 1984–1993

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Differentially regulated miRNAs in ischemic stroke
patients with different conditions
M. Vijayan, P.H. Reddy . Peripheral biomarkers of stroke: Focus on circulatory microRNAs . 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

18. 2- Biomarkers of inflammation
Interleukin-6
 Released in the post-stroke setting and serves mediators
in the acute phase of AIS
 Interleukin-6 (IL-6) expression was upregulated
following brain ischemia when measured in the first 24
h in the peripheral blood. .
 IL-6 rise in peripheral blood had
a neuroprotecive effect and was
associated with better outcome,
but showed no value
as a diagnostic tool
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19. Procalcitonin level
• Significantly increased in ischemic stroke patients when
compared to a normal control group.
• Patients with procalcitonin level above 1.20 ng/ml were
at a higher risk for AIS in comparison to healthy
individuals
Tumor necrosis factor-a (TNF-a)
• Tumor necrosis factor-a reaches a high concentration in
the first 6 h after AIS, but its similar behavior in other
inflammatory and infectious processes limits its capacity
to be used as a valuable diagnostic tool.
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20. 3-Biomarkers of oxidative damage
Thioredoxin
A redox-regulating protein with antioxidant activity that
can be a potential indicator of oxidative stress in strokes
Oxidative stress index (OSI)
 The ratio of total oxidant status (TOS ) level to total
antioxidant status (TAS) level.
 Serum thioredoxin level was higher in patients with AIS
when compared to healthy patients (15.03 ng/ml vs.
8.95 ng/ml)
 Strong correlation between increased thioredoxin serum
levels and risk of AIS
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21.  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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22. 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

25. • A panel of nine apo-lipoproteins was tested as a
tool to distinguish IS and ICH patients within the
first week after symptom onset using a mass
spectrometry assay.
• Apo C-I and Apo C-III reported to provide the
best classification power as individual markers
but combining Apo C-III and Apo A-I provided
the best discrimination overall .
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26. 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 ?

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Dagonnier M, Donnan GA, Davis SM, Dewey HM and Howells DW (2021) Acute Stroke Biomarkers: Are We There Yet?
Front. Neurol. 12:619721. doi: 10.3389/fneur.2021.619721
Main clinical uses and their linked potential biomarkers.

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Dagonnier M, Donnan GA, Davis SM, Dewey HM and Howells DW (2021) Acute Stroke Biomarkers: Are We There Yet?
Front. Neurol. 12:619721. doi: 10.3389/fneur.2021.619721

31. 5:20 AM 31
Dagonnier M, Donnan GA, Davis SM, Dewey HM and Howells DW (2021) Acute Stroke Biomarkers: Are We There Yet?
Front. Neurol. 12:619721. doi: 10.3389/fneur.2021.619721

32. Conclusions
 Improving in patient outcomes in acute stroke
requires a rapid and accurate diagnosis of stroke and
its subtypes.
 A biomarker that could differentiate between
hemorrhagic and ischemic stroke or risk of
subsequent bleeding would, in theory, permit
widespread initiation of thrombolysis in the
ambulance and save valuable time and brain tissue.
 The ultimate aim of the stroke biomarker research is
the development of a point of care device. A quick
and reliable bedside biomarker assessment would
revolutionize the acute stroke management.
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33. THANK YOU
PROF./MOUSTAFA RIZK
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