stroke biomarkers a new era with diagnostic promise

1. Clinical Chemistry
Bio Markers of Brain Injury

2. Stroke Biomarkers : a new era with
diagnostic promise ?
By
Prof. Moustafa Rizk
Prof. of Clinical Pathology
Faculty of Medicine, University of Alexandria
7/6/2023
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3. 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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4. Stroke Types
 80% ischemic
 Thrombosis
 Embolism
 Hypoperfusion
 20% hemorrhagic
 Intracerebral
 Subarachnoid
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5. 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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6. 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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7. 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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8. 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 use
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9.  Subarachnoid hemorrhage (SAH)
–Result from rupture of
berry aneurysm or
(rupture of AVMs)
Hemorrhagic Stroke
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10. A transient ischaemic attack (TIA)
Is defined as ‘stroke symptoms and signs that resolve
within 24 hours’
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11. 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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12. 5:54 PM 12
Sources of the major candidate biomarkers.

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 Differentiate hemorrhagic and ischemic stroke .
 Predict stroke prognosis,
 Facilitate therapeutic stratification
 Therapeutic monitoring,
 Indicating risk of hemorrhagic transformation
after stroke or after rt-PA treatment.
 Act as a “stroke clock” to aid in assessing time
of stroke onset to increase the number of IS
able to benefit from treatment with rt-PA
 Aid early identification and pre-hospital
stratification of ischemic stroke patients.
An ideal stroke biomarker)

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 Facilitate directing the patient to a hospital
where thrombectomy is performed without
losing crucial time by performing brain
imaging in the nearest hospital and then
transferring the patient to the comprehensive
stroke center.
 It is known that substantial delays of 120 min.
are associated with secondary transfer vs. the
direct approach.

15. 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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16. 5- Biomarkers of thrombus formation
Ex. D-dimers
Platelet reactivity
6- Biomarkers of cardiac function
Ex. N-terminal pro-Brain Natriuretic Peptide
High sensitivity troponin T
7- Biomarkers of stroke risk
Ex.
-Lipoprotein a ssociated phospholipase A2 (Lp-PLA2)
-High levels of oxidized low-density Lipoproteins
-Asymmetric dimethylarginine (ADMA)
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17. S100β
S100B, a glial protein, highly specific to nervous tissue, was one
of the first molecules suggested as a candidate to aid IS diagnosis
 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

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 S100 peaks from symptom onset between 24 h
and 120 h with significantly raised values
measured from 0 to 120 h.
 Higher S100 values indicated significantly larger
infarction volumes, more severe strokes, and
worse functional outcome.
 There was a significant difference in S100 levels
between AIS patients and controls

19. 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 intracerebral hemorrhage in patients treated
with thrombolytic therapy
• 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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20. 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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21. NSE
 Significantly raised in stroke patients compared to controls
and 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 NIH Stroke Scale (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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22. 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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23. • 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

24. • 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.
Circulating MicroRNAs as Novel Potential Biomarkers for Early Diagnosis of Acute Stroke in Humans. Journal of
Stroke and Cerebrovascular Diseases, Vol. 23, No. 10 (November-December), 2014: pp 2607-2613

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Elevated hsa-miR-106b-5P and hsa-miR-4306 and decreased hsa-
miR-320e and hsa-miR-320d in plasma may be novel biomarkers for
the early detection of acute stroke in humans.
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

28. 2- Biomarkers of inflammation
Interleukin-6
 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 ssociated with better outcome, but showed no
value as a diagnostic tool.
 IL-6 serum level showed a significant inverse correlation
with both final neurological impairment and infarct size.
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29. 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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30. 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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31.  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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32. 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

35. • 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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36. 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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37. 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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Data show that FABP4 is a novel independent prognostic marker
improving the currently used risk stratification of stroke patients.
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
Tu WJ, Zeng XW et al.. Circulating FABP4 (Fatty Acid-Binding Protein 4) Is a Novel Prognostic Biomarker in Patients
With Acute Ischemic Stroke. Stroke. 2017 Jun;48(6):1531-1538.

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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

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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

42. 5:54 PM 42
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.
These results suggest that information
obtained from a 5-biomarker panel may
add valuable information in the early
evaluation and management
of patients with stroke-like symptoms.

43. 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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44. PROF./MOUSTAFA RIZK