Biochemical Markers of Brain Injury and Inflammation

Biochemical Markers of Brain
Injury and Inflammation
Dr Roy Sherwood
Department of Clinical Biochemistry
King’s College Hospital
London, UK
Why do we need biochemical markers of
brain injury and inflammation?
• Stroke
• Closed head injury
• Sub arachnoid haemorrhage
• Diagnosis? - probably not

• Determination of severity? - yes
• Assessment of prognosis? – yes
• Monitoring of course and therapy? - yes
What markers are available?
• Protein S100B
• Neurone specific enolase
• Glial acidic fibrillary protein
• Inflammatory markers
What are S100 proteins ?
S100 is a family of low molecular weight (9-13kDa) Ca2+-

binding proteins
Named S100 protein on the basis of its solubility at neutral pH
in a 100% saturated ammonium sulphate
Around 1978 it was found that S100 consisted of two closely

related molecules S100 beta (b, b, B) and S100 alpha (a, a,
A1) and believed to exist in three isoforms: S100aa, S100ab,
S100bb
To date the S100 protein family contains about 20 members

S100B
S100B is the first known member of the S100

protein family
Within cells, S100B exists as a homodimer of

S100B2 (S100bb, S100b) or as a heterodimer of
S100A1B (S100ab)
Dimerization of S100B is important for the

biological activity
Dimer structure of S100B protein
Schematic representation of the assumed mode of
interaction of an S100B dimer with a target protein
Tissue distribution of S100B
S100B is found primarily in glial cells and astrocytes in the

central nervous system (CNS) and Schwann cells in the
peripheral nervous system.
Outside the nervous system:

Adipose tissue
Chondrocytes
Melanocytes
S100B distribution
Tissue Concentration
Brain 100 %
Adipose tissue 2.8-5.6 %
Rectum 3.4 %
Skin 2.0 %
Oesophagus 1.5 %
Stomach 1.5 %
Pancreas 1.3 %
Heart 0.6 %
Skeletal Muscle 0.2 %
Lung 0.1 %
Kidney 0.04 %
Liver 0.02 %
Differential distribution of S100B in different tissues (Haimoto et al and Kato et al)
Concentration expressed as portion of total cell protein content where brain has been set to 100 %
Functions of S100B
• Regulates Ca 2+ homoeostasis signal transduction via cAMP and

by interaction with a number of neuronal protein kinase C
substrates
• Inhibits protein phosphorylation
• Regulates energy metabolism and enzyme activity
• S100B regulates the activity of Ndr (a nuclear-serine/threonine
protein kinase) which is involved in cell division (mitogenic
activity)
• S100B regulates cell growth and proliferation.
• S100B regulates cell structure - involved in microtubule assembly
• Regulates target proteins that possess a peptide sequence of (R/K)
(L/I) (XWXXIL) called TRTK-12
Measurement of serum S100B
IRMA (Sangtec)
‘I125” as a tracer
LIA-mat Sangtec S100 – semi automated

Chemiluminescence – isoluminol derivatives as a tracer
Automated analysers – Chemiluminescence:
DiaSorin Liaison
Serum S100B level – age dependency
Portela et al. (2002)

Clinical applications of Biochemical Brain
Markers
Central nervous system – marker of the extent of

brain damage:
Stroke
Cardiac arrest/surgery
Head injury etc.
S100 in Strokes
• S100 measured in CSF has been found to be raised

following traumatic brain injury and haemorrhagic
strokes
• One previous report of serum S100 in neurological
disorders found S100 to be raised in 90% of stroke
patients as well as in patients with meningitis and
CNS tumours
Patients and Methods
• Samples for S100 were collected from 81 patients admitted

to an acute stroke unit. Samples were taken within 48h
post-ictus in all patients. Further samples were taken at 3
months in 57 surviving patients. Samples were also taken
from 51 age, gender ethnically matched control subjects.
• Clinical outcome was assessed using functional measures

of disability, the Barthel index, handicap, the Rankin scale
and the dependence scale of Lindley.
Results
Control IS HS p
Age (y) 71.3 (7.3) 72.7 (7.7) 70.9 (6.7) 0.73
Gender 41 60 46 0.11
(% male)
Race (% 62 75 54 0.30
Caucasian)
S100 0.11 0.27 0.43 <0.0001
(g/L) (0.03) (0.09) (0.23)
Infarct size
TACI PACI LACI POCI p
n 15 20 24 9
S100 0.45 0.27 0.20 0.25 0.0005

(g/L) (0.22) (0.07) (0.06) (0.25)
Discharge destination
Died Institution Home
S100 0.63 0.37 0.26

(g/L) (0.25) (0.13) (0.11)
Outcome measures
S100 v Spearman rank rs p
Barthel -0.285 0.01
Rankin 0.313 0.004
Lindley 0.262 0.018

Conclusions
• Serum S100 rises in the first 24 h following a

stroke, slowly falling to normal over 3 months
• The S100 concentration in the first 48 h is
positively correlated to infarct size.
• S100 on admission is related to cognitive function
at 3 months
Hypoglycaemic comas
• There are suggestions that hypoglycaemic comas

may result in permanent neurological sequelae
• S100 was measured in 16 patients with IDDM

immediately following an episode of severe
hypoglycaemia and at 9 days and in 10 matched
IDDM normoglycaemic controls
Results
• S100 in hypoglycaemic patients was 0.05 (0.01)

g/L immediately following the attack and 0.05
(0.01) g/L after 9 days and in controls 0.05 (0.01)
g/L.
• In one patient who died of cerebral oedema and
hypoglycaemia due to an insulin overdose the
serum S100 was 3.9 g/L. S100 increased in
advance of clinical or radiological signs of
cerebral oedema.
Out of Hospital Cardiac arrest
In-hospital death, and predicting it...
90
80
70
M o rtality (%)
60
50
40
30
20
10
0
3 4 to 5 6 to 1 2 1 3 to 1 5
A d m is s io n G C S
Grubb et al, Lancet 1995;346:417-421.

Clinical Problem Two
Cognitive dysfunction in survivors
25
P o s t-A rre s t
20
N u m b er o f cases
C o n tro ls
15
10
5
0
N o rm a l M ild M o d e ra te S e v e re
R iv e rm e a d G ro u p in g
Grubb et al, BMJ 1996;313:143-146.

Importance of Prognostic Assessment
•assists decision-making regarding:

• institution of new treatment (e.g. ventilation,
enteral feeding, cardiac assist)
• resuscitation status
• where to manage the patient
•assist in counselling relatives
•helps direct resources where most needed
Sensitivity or Specificity
Which is more important ?
• can’t have a test that’s 100% sensitive for in-

hospital death as there are so many modes of death
• arrhythmia, pump failure, sepsis, sequelae of
brain injury, etc.
• what is important is being beyond reasonable doubt
when patient’s prognosis is hopeless
• specificity is what really matters
Markers of Brain Injury
NSE and S-100
• Neuron-specific enolase is neuron derived, but also

found in RBCs
• S-100 is glial protein. A previous study in cardiac
arrest* suggested cut-off of 0.2 g/L at 24 hours
predicted a hopeless prognosis
* Rosén et al. Stroke 1998;29:473-7.

Objectives
• to determine whether brain enzymes are

useful predictors of mortality after OHCA
• to verify whether S-100 > 0.2 g/L really
predicts a hopeless prognosis
• to determine whether the assays are
sensitive enough to predict cognitive
impairment
Methods
• 85 consecutive OO-HA victims entered into

study
• blood drawn for NSE & S-100 on
admission, at 24-48 hrs, and 72-96 hrs
• in survivors, memory assessed within 24 hrs
of discharge using Rivermead Memory test
Baseline Characteristics
Age (years) 68.1 (range 32.2 to 93.2)

M/F 58 / 27
PREVIOUS MEDICAL HISTORY

Myocardial infarction 40 (47%)
Angina 38 (45%)
Heart failure 25 (29%)
Hypertension 24 (28%)
Arrhythmia 28 (33%)
Valve disease 13 (15%)
Any cardiovascular condition 60 (80%)

S-100 and survival
1 .2 p<0.001
S U R V IV E D
D IE D
1
S -1 0 0 (m c g /l)
p<0.001
0 .8
p=0.003
0 .6
0 .4
0 .2
0
A d m is s io n 2 4 -4 8 h 7 2 -9 6 h
NSE and survival
40 S u rvived
p<0.001
D ied
35
30
NSE (m cg /l)
25 p=0.001
20 p<0.001
15
10
5
0
A d m issio n 24-48 h 72-96 h
S-100 at 24-48 hours
S-100 co n cen tratio n (m cg /l)
10
0
0 1 2 3 4
S URV IV E D D IE D
S-100 at 24-48 hours (log transformed)
10
lo g S-100 co n cen tratio n
1
0 1 2 3 4
0.1
0.01
0.001
S URV IV E D D IE D
NSE at 24-48 hours
180
160
NSE co n cen tratio n
140
120
100
80
60
40
20
0
0 1 2 3 4
S URV IV E D D IE D
NSE at 24-48 hours (log transformed)
1000
lo g NSE co n cen tratio n
100
10
1
0 1 2 3 4
S URV IV E D D IE D
• six patients survived with a 24 hour S-100 level
greater than the threshold concentration of 0.2 g/L
• the cut-off S-100 value which gave 100%

specificity for in-hospital death was 0.6 g/L
S-100 and memory
Spearman’s rho significance
ADMISSION -0.498 p=0.002
24-48 HRS -0.486 p=0.003*
72-96 HRS -0.441 p=0.009
* threshold value of 0.32 g / L predicts memory impairment with 100%

specificity
NSE and memory
Spearman’s rho significance
ADMISSION -0.099 p=0.570
24-48 HRS -0.357 p=0.035*
72-96 HRS -0.278 p=0.111

OHCA Conclusions
• NSE and S-100 concentrations are significantly raised in

resuscitated OHCA victims who subsequently die in
hospital
• the optimum cut-off value for S-100 was 0.6 g /L
• a S-100 concentration > 0.32 g /L at 24 hours is a

powerful predictor of cognitive deficit
• brain marker assays may assist prognostic assessment of

cardiac arrest victims
POCT Stroke testing
• Biosite have launched POCT stroke panel for the Triage –

analytes include:
– S100B
– BNP
– TNFalpha
– VWF
• The results are put together in an algorithm which gives a
probability that the patient has had a stroke.
Overall conclusions
Biochemical brain markers are coming of age
but apart from S100B it is not yet clear
which will be of most use. Panels of
markers are the most likely way forward.
POCT methods may be required in minor
head injury applications as the time window
is narrow.
For the interested !
The 5th International Conference on
Biochemical Brain Markers
May 11-14 2009
Lund, Sweden
Abstract deadline: 31st March 2009

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Biochemical Markers of Brain

Injury and Inflammation

• Diagnosis? - probably not

S100 is a family of low molecular weight (9-13kDa) Ca2+-

Around 1978 it was found that S100 consisted of two closely

To date the S100 protein family contains about 20 members

S100B is the first known member of the S100

Within cells, S100B exists as a homodimer of

Dimerization of S100B is important for the

S100B is found primarily in glial cells and astrocytes in the

Outside the nervous system:

• Regulates Ca 2+ homoeostasis signal transduction via cAMP and

LIA-mat Sangtec S100 – semi automated

Portela et al. (2002)

Central nervous system – marker of the extent of

• S100 measured in CSF has been found to be raised

• Samples for S100 were collected from 81 patients admitted

• Clinical outcome was assessed using functional measures

Age (y) 71.3 (7.3) 72.7 (7.7) 70.9 (6.7) 0.73

TACI PACI LACI POCI p

S100 0.45 0.27 0.20 0.25 0.0005

Died Institution Home

S100 0.63 0.37 0.26

S100 v Spearman rank rs p

Barthel -0.285 0.01

Rankin 0.313 0.004

Lindley 0.262 0.018

• Serum S100 rises in the first 24 h following a

• There are suggestions that hypoglycaemic comas

• S100 was measured in 16 patients with IDDM

• S100 in hypoglycaemic patients was 0.05 (0.01)

Grubb et al, Lancet 1995;346:417-421.

Grubb et al, BMJ 1996;313:143-146.

•assists decision-making regarding:

• can’t have a test that’s 100% sensitive for in-

• Neuron-specific enolase is neuron derived, but also

* Rosén et al. Stroke 1998;29:473-7.

• to determine whether brain enzymes are

• 85 consecutive OO-HA victims entered into

Age (years) 68.1 (range 32.2 to 93.2)

PREVIOUS MEDICAL HISTORY

Any cardiovascular condition 60 (80%)

• the cut-off S-100 value which gave 100%

Spearman’s rho significance

ADMISSION -0.498 p=0.002

24-48 HRS -0.486 p=0.003*

72-96 HRS -0.441 p=0.009

* threshold value of 0.32 g / L predicts memory impairment with 100%

Spearman’s rho significance

ADMISSION -0.099 p=0.570

24-48 HRS -0.357 p=0.035*

72-96 HRS -0.278 p=0.111

• NSE and S-100 concentrations are significantly raised in

• the optimum cut-off value for S-100 was 0.6 g /L

• a S-100 concentration > 0.32 g /L at 24 hours is a

• brain marker assays may assist prognostic assessment of

• Biosite have launched POCT stroke panel for the Triage –

Abstract deadline: 31st March 2009