Research
Original Investigation
Blood Biomarkers for Brain Injury
in Concussed Professional Ice Hockey Players
Pashtun Shahim, MD; Yelverton Tegner, MD, PhD; David H. Wilson, PhD; Jeffrey Randall, PhD;
Tobias Skillbäck, MD; David Pazooki, MD, PhD; Birgitta Kallberg, BSc; Kaj Blennow, MD, PhD;
Henrik Zetterberg, MD, PhD
Editorial
IMPORTANCE Lack of objective biomarkers for brain damage hampers acute diagnosis and
clinical decision making about return to play after sports-related concussion.
Supplemental content at
jamaneurology.com
OBJECTIVES To determine whether sports-related concussion is associated with elevated
levels of blood biochemical markers of injury to the central nervous system and to assess
whether plasma levels of these biomarkers predict return to play in professional ice hockey
players with sports-related concussion.
DESIGN, SETTING, AND PARTICIPANTS Multicenter prospective cohort study involving all 12
teams of the top professional ice hockey league in Sweden, the Swedish Hockey League. Two
hundred eighty-eight professional ice hockey players from 12 teams contesting during the
2012-2013 season consented to participate. All players underwent clinical preseason baseline
testing regarding concussion assessment measures. Forty-seven players from 2 of the 12 ice
hockey teams underwent blood sampling prior to the start of the season. Thirty-five players
had a concussion from September 13, 2012, to January 31, 2013; of these players, 28
underwent repeated blood sampling at 1, 12, 36, and 144 hours and when the players
returned to play.
MAIN OUTCOMES AND MEASURES Total tau, S-100 calcium-binding protein B, and
neuron-specific enolase concentrations in plasma and serum were measured.
RESULTS Concussed players had increased levels of the axonal injury biomarker total tau
(median, 10.0 pg/mL; range, 2.0-102 pg/mL) compared with preseason values (median, 4.5
pg/mL; range, 0.06-22.7 pg/mL) (P < .001). The levels of the astroglial injury biomarker S-100
calcium-binding protein B were also increased in players with sports-related concussion
(median, 0.075 μg/L; range, 0.037-0.24 μg/L) compared with preseason values (median,
0.045 μg/L; range, 0.005-0.45 μg/L) (P < .001). The highest biomarker concentrations of
total tau and S-100 calcium-binding protein B were measured immediately after a
concussion, and they decreased during rehabilitation. No significant changes were detected
in the levels of neuron-specific enolase from preseason values (median, 6.5 μg/L; range,
3.45-18.0 μg/L) to postconcussion values (median, 6.1 μg/L; range, 3.6-12.8 μg/L) (P = .10).
CONCLUSIONS AND RELEVANCE Sports-related concussion in professional ice hockey players
is associated with acute axonal and astroglial injury. This can be monitored using blood
biomarkers, which may be developed into clinical tools to guide sport physicians in the
medical counseling of athletes in return-to-play decisions.
Author Affiliations: Author
affiliations are listed at the end of this
article.
JAMA Neurol. doi:10.1001/jamaneurol.2014.367
Published online March 13, 2014.
Corresponding Author: Pashtun
Shahim, MD, Clinical Neurochemistry
Laboratory, Sahlgrenska University
Hospital, Mölndal,
S-431 80 Mölndal, Sweden
([email protected]).
E1
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Research Original Investigation
Blood Biomarkers for Brain Injury in Hockey
C
oncussion in athletes practicing contact sports such as
ice hockey, American football, and boxing is a growing
problem worldwide.1-3 The term is often used interchangeably with mild traumatic brain injury (TBI). During recent years, many professional athletes have retired owing to
chronic symptoms after repeated concussions.4 An international consensus statement defined concussion as a complex
pathophysiological process affecting the brain, induced by traumatic biomechanical forces.5 Mild concussion causes no loss
of consciousness, but many other symptoms such as dizziness, nausea, reduced attention and concentration, memory
problems, and headache may occur. More severe concussion
also causes unconsciousness. The pathophysiology underlying concussion is an acute disturbance of neuronal function
together with damage to neuronal and glial cells, which may
have both subacute and chronic consequences.6 Most concussions resolve within days to weeks, but 10% to 15% remain
symptomatic more than 1 year after injury.7,8 The danger of developing chronic or even progressive symptoms has been linked
to repeated concussions before the brain has recovered
properly.4 Tools to detect and monitor injury to the central nervous system (CNS) due to concussion are therefore of great importance and could objectively inform decisions on when return to play (RTP) may be safe.
Cerebrospinal fluid (CSF) is in direct contact with the brain
parenchyma and thus is a suitable biofluid to monitor biochemical changes in the CNS. Elevated levels of CSF biomarkers of axonal damage, eg, total tau (T-tau) and neurofilament
light (NFL), are found after acute damage to the brain such as
stroke and subarachnoid hemorrhage, and the levels of these
biomarkers correlate with the severity of the brain damage.9,10
Cerebrospinal fluid T-tau has been measured in severe TBI, and
its concentrations correlate with 1-year outcome.11,12 In the context of contact sports, CSF NFL and T-tau have been shown to
increase in amateur boxers, even after bouts without any
knockout.13,14 Further, the CSF levels of NFL and T-tau correlate with the number and severity of received head blows and
return to normal levels after a period of rest from boxing.13,14
These findings indicate that CSF biomarkers may be used to
identify brain damage in concussed athletes and as a guide for
medical teams in RTP decisions.15 However, owing to the invasive nature of lumbar puncture, these results may be difficult to implement in the routine evaluation and follow-up of
concussed athletes. Thus, analysis of brain injury biomarkers
in blood would be preferable. Neuron-specific enolase (NSE)
is a biomarker for neuronal injury, and an increase in serum
levels of NSE was found in boxers who received repetitive
trauma to the head.16 Similarly, increases in NSE and the glial
cell biomarker S-100 calcium-binding protein B (S-100B) were
found in serum of amateur boxers who received direct punches
to the head compared with boxers who received body
punches.17 Last, using a novel ultrasensitive method to measure tau in plasma,18 increased plasma tau concentrations were
found in Olympic boxers after bout, with normalization of levels after a resting period.19
The diagnosis of sports-related concussion is based on the
evaluation of subjective symptoms after exclusion of overt
gross structural brain damage. Thus, reliable biomarkers to asE2
sess TBI and recovery in concussed athletes are highly desirable. In this study, we examined concentrations of plasma Ttau and serum S-100B and NSE in professional ice hockey
players who had a sports-related concussion. Postconcussion
levels of T-tau, NSE, and S-100B were compared with preseason levels in a cohort of hockey players prior to the start of
the hockey season.
Methods
Study Population
This is a prospective cohort study of concussion among professional ice hockey players from the Swedish Hockey League
who competed during the first half of the 2012-2013 ice hockey
season. The Swedish Hockey League is the top professional ice
hockey league in Sweden with 12 teams, each consisting of 24
players. The study was approved by the Ethics Committee for
Medical Research at the University of Gothenburg and by the
Swedish Ice Hockey Association. Written informed consent was
obtained from all participants. The teams’ physicians were
present at all regular season games, documenting signs and
symptoms of concussion and physical examination findings
in the event of a concussion. Physicians also recorded the date
when a player had completely recovered from his concussion
and was able to return to unrestricted competition. During the
preseason, the team physicians were provided with a concussion kit containing an injury protocol, the Rivermead Post Concussion Symptoms Questionnaire,20 instructions for blood
tests, blood sampling equipment and tubes, and instructions
for the local laboratory to handle blood samples.
All players were examined physically and with the Standardized Assessment of Concussion21,22 prior to the start of the
season. Players from 2 of the contesting teams, Frölunda
Hockey Club and Luleå Hockey, were sampled for baseline values prior to the start of ice hockey season and before the players went on ice. Players from Luleå Hockey were also sampled
immediately after a friendly game without concussion incidents. In players who sustained head injury or concussion during the season, consecutive blood samples were collected at
1, 12, 36, and 48 hours as well as 144 hours after the injury or
the date on which the player returned to unrestricted competition. The diagnosis of concussion was made according to the
latest diagnostic guidelines on sports-related concussion, and
players with concussion were managed according to these
guidelines.23,24
Biochemical Procedures
Blood samples were collected by venipuncture into gelseparator tubes for serum and ethylenediaminetetraacetic acid
(EDTA) tubes for plasma and centrifuged within 20 to 60 minutes. Serum and plasma were separated, aliqouted, and stored
at −80°C pending biochemical analysis. Samples for S-100B and
NSE were analyzed on a Modular E170 instrument (Roche Diagnostics) with reagents from the same manufacturer. Plasma
T-tau was measured with a novel immunoassay using digital
array technology (Quanterix Corp) as previously described.25
The limit of detection for the assay is 0.02 pg/mL, which is more
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Blood Biomarkers for Brain Injury in Hockey
Original Investigation Research
Figure 1. Preseason and Postconcussion Levels of Total Tau (T-tau), S-100 Calcium-Binding Protein B (S-100B), and Neuron-Specific Enolase (NSE)
P =.002
A
100
10
1
0.1
0.01
Serum NSE, μg/L
100
0.1
0.01
0.001
Preseason
Postconcussion
P =.10
C
1
Serum S-100B, μg/L
Plasma T-tau, pg/mL
P =.40
B
1000
10
1
0.1
0.01
Preseason
A, Plasma T-tau was higher in postconcussion samples (all times) compared
with preseason samples. There were no significant changes in the serum levels
Postconcussion
Preseason
Postconcussion
of S-100B (B) and NSE (C) after concussion compared with preseason samples.
Horizontal lines indicate medians.
Table 1. Demographic Data and Blood Concentrations of Total Tau, Neuron-Specific Enolase, and S-100
Calcium-Binding Protein B
Median (Range)
Characteristic
Age, y
Preseason
(n = 47)
28 (19-38)
Postconcussiona
(n = 28)
P Value
27 (19-40)
.40
10.0 (2.0-102)
<.001
T-tau, pg/mL
4.5 (0.06-22.7)
S-100B, μg/L
0.045 (0.005-0.45)
0.075 (0.037-0.24)
<.001
6.1 (3.6-12.8)
6.5 (3.45-18.0)
.20
Abbreviations: NSE, neuron-specific
enolase; S-100B, S-100
calcium-binding protein B; T-tau, total
tau.
a
NSE, μg/L
than 1000-fold more sensitive than conventional immunoassays for the protein. The assay uses Tau 5 monoclonal antibody for capture (Covance) and HT7 and BT2 monoclonal antibodies for detection (Thermo Scientific Pierce). This
combination of antibodies reacts with both normal and phosphorylated tau with epitopes in the midregion of the molecule, making the assay specific for all tau isoforms. All samples
were analyzed at the same time using the same batch of reagents by board-certified laboratory technicians who were
blinded to clinical information. Intra-assay coefficients of variation were below 3% for S-100B and NSE and 11.5% for T-tau.
Statistical Analysis
For the paired observations, the Wilcoxon signed rank test was
used. For the TBI vs preseason comparison, the MannWhitney U test was used, and for multiple group comparisons, the Kruskal-Wallis test was used. The Spearman rank correlation coefficient (ρ) was used for analyses of correlation
between changes in various biomarker levels after concussion. Linear regression analysis examined the prediction of concussion severity by postconcussion biomarker levels. The cubic spline interpolation for graphical representation of the
biomarker changes was conducted with SPSS version 20.0 statistical software (IBM Corp). The area under the receiver operating characteristic curve (AUC) was calculated for all biomarkers in concussed players vs biomarker changes after a
friendly game. Statistical significance was determined at
P < .05. All statistical calculations were performed using SPSS
version 20.0 and GraphPad Prism version 5.0 (GraphPad Inc).
The biomaker values are from 1 hour
after concussion.
Results
Of a total 288 professional ice hockey players, 35 had a sportsrelated concussion between September 13, 2012, and January
31, 2013. We included 28 of these players in the study. The remaining concussed players either declined to participate or had
an uncertain diagnosis of concussion. Three of the included
players had concussion with unconsciousness and 25 experienced symptoms such as headache, confusion, dizziness, or
nausea. Many players were free from symptoms less than 6 days
after injury, but in 15 players the symptoms lasted longer than
6 days. The players’ postconcussion biomarker results were
compared longitudinally as well as with preseason values from
47 players from 2 of the competing teams. There were no correlations between biomarker levels at baseline and age (eFigure in Supplement).
The T-tau levels were significantly higher in postconcussion samples (all times) compared with preseason samples
(P = .002) (Figure 1A). There was no significant increase in
the levels of S-100B and NSE in postconcussion samples (all
times) compared with preseason samples (Figure 1B and C).
However, the levels of both T-tau (P < .001) and S-100B
(P < .001) were higher immediately after a concussion compared with preseason samples (Table 1). There was no significant difference in the levels of NSE (Table 1). Further, the
levels of S-100B and NSE, but not T-tau, increased after a
friendly game without concussion compared with baseline
(eTable in Supplement).
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Research Original Investigation
Blood Biomarkers for Brain Injury in Hockey
Figure 2. Total Tau (T-tau), S-100 Calcium-Binding Protein B (S-100B),
and Neuron-Specific Enolase (NSE) Measured at Selected Times
in Concussed Hockey Players
A
200
Plasma T-tau, pg/mL
150
100
50
50
25
0
Severity of Concussion
1
12
36
144
RTP
Preseason
RTP
Preseason
Time After Concussion, h
B
Serum S-100B, μg/L
0.3
0.2
0.1
0
1
12
36
144
Time After Concussion, h
C
25
Serum NSE, μg/L
20
15
10
5
0
1
12
36
144
RTP
Preseason
Time After Concussion, h
A, Concentrations of T-tau peaked during the first hour after concussion. The
levels declined significantly (P < .001) during the first 12-hour period. Further
declines in T-tau between 12 and 144 hours were not statistically significant
(P = .15). Concentrations of T-tau remained elevated 144 hours after concussion
compared with preseason levels (P = .002). B, Similar to T-tau, the levels of
S-100B peaked during the first hour after concussion and declined during the
first 12-hour period (P < .001). C, Concentrations of NSE remained unchanged
over time after concussion. RTP indicates return to play.
E4
The highest levels of T-tau were measured during the first
hour after concussion. These levels declined during the first
12-hour period (P < .001) (Figure 2A). Further declines between 12 and 144 hours in T-tau were not statistically significant (P = .15), and the level of T-tau remained elevated 144
hours after concussion compared with preseason samples
(P = .002). There was a delayed increase in T-tau between 12
and 36 hours after concussion, with a trend for a second T-tau
peak at 36 hours after concussion (Figure 3A).
Similar to T-tau, the levels of S-100B peaked during the first
hour after concussion and declined during the first 12-hour period (P < .001) (Figure 2B). However, unlike T-tau, we detected no delayed elevation of S-100B between 12 and 36 hours
after concussion (Figure 3B). The levels of NSE remained unchanged over time in concussed players (Figure 2C and
Figure 3C).
Assessing the severity of TBI is important in the management
of sports-related concussion. Recently, the emphasis has been
on the time it takes until a player is declared fit to return to unrestricted competition.24,26 We graded concussions according to the latest guidelines,24 which are based on the resolution of concussion symptoms. We graded the players with
concussion into 4 categories: (1) those who were free from
symptoms and had safe RTP within 6 days after a concussion
(n = 12), (2) those who had safe RTP 7 to 10 days after concussion (n = 9), (3) those who had safe RTP more than 10 days after concussion (n = 7), and (4) those who also had loss of consciousness due to the head impact (n = 3). The level of T-tau 1
hour after concussion was not statistically significantly different between the concussion categories; however, there were
trends toward higher levels in players who had symptoms for
more than 10 days or had loss of consciousness (Figure 4A).
The levels of S-100B 1 hour after concussion were significantly higher in players with loss of consciousness (P < .05) and
those with RTP more than 10 days after concussion (P < .01)
than in players with concussion symptoms resolving within
6 days (Figure 4B). There was no significant difference in the
levels of NSE at 1 hour after concussion between these different concussion categories (Figure 4C).
The concentration of T-tau 1 hour after concussion correlated with the number of days it took for concussion symptoms to resolve (ρ = 0.60; 95% CI, 0.23 to 0.90; P = .002)
(Figure 4A). Similar to T-tau, there was a significant correlation between the concentration of S-100B and the time to resolution of concussion symptoms (ρ = 0.75; 95% CI, 0.50 to 0.90;
P < .001) (Figure 4B). The concentration of NSE did not correlate with the resolution of concussion symptoms (ρ = 0.20; 95%
CI, −0.20 to 0.55; P = .30) (Figure 4C). Using linear regression,
only T-tau concentrations at 1 hour after concussion predicted the number of days it took for the concussion symptoms to resolve and the players to return to unrestricted competition (Figure 4D-F).
As postconcussion sampling at 1 hour may not always be
practical, we examined the relationship between biomarker elevation 144 hours after concussion and persistence of postconcussive symptoms (PCS). The levels of T-tau 144 hours
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Blood Biomarkers for Brain Injury in Hockey
Original Investigation Research
Figure 3. Biomarker Concentration Time Course Profile in Concussed Hockey Players
A
10 000.0
Plasma log(T-tau), pg/mL
1000.0
100.0
10.0
1.0
0.1
Preseason
30
80
130
180
130
180
130
180
Time, h
B
Serum log(S-100B), μg/L
1.000
0.100
0.010
0.001
Preseason
30
80
Time, h
C
100.00
Serum log(NSE), μg/L
10.00
1.00
0.10
0.01
Preseason
30
80
Time, h
The spline curves represent the concentration time course of total tau (T-tau)
(A), S-100 calcium-binding protein B (S-100B) (B), and neuron-specific enolase
(NSE) (C) in relation to preconcussion levels, estimated on the basis of
preseason values from 2 complete teams, from 1 hour after concussion until
players return to unrestricted competition. The vertical lines represent the time
of brain injury.
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E5
Research Original Investigation
Blood Biomarkers for Brain Injury in Hockey
Figure 4. Biomarker Concentrations in Relation to Severity of Brain Injury
A
B
80
40
0
20
Serum NSE , μg/L
0.15
Serum S-100B, μg/L
Plasma T-tau, pg/mL
120
C
0.10
0.05
0.00
≤6
7-10
>10
≤6
7-10
Unconscious
≤6
50
25
0
15
20 20 60 100
>10
Unconscious
R2 = .0016; P = .85
F
20
Serum NSE, μg/L
100
10
7-10
PCS, d
0.3
Serum S-100B, μg/L
Plasma T-tau, pg/mL
>10
R2 = 0.0033; P = .08
E
200
5
5
PCS, d
R2 = 0.34; P = .002
0
10
0
Unconscious
PCS, d
D
15
0.2
0.1
0
15
10
5
0
0
Postconcussion, d
5
10
15
20 20 60 100
0
5
Postconcussion, d
Total tau (T-tau) (A), S-100 calcium-binding protein B (S-100B) (B), and
neuron-specific enolase (NSE) (C) concentrations 1 hour after concussion are
shown in relation to concussion severity. Values are presented as means; error
bars indicate standard error of mean; and PCS indicates postconcussive
10
15
20 20 60 100
Postconcussion, d
symptoms. Concentrations of T-tau (D), S-100B (E), and NSE (F) 1 hour after
concussion are shown in relation to resolution of PCS and return to play. One
player returned to play 69 days after concussion. Another resigned owing to
persistent PCS and was entered as more than 90 days in the graph.
Table 2. Blood Concentrations of Total Tau, Neuron-Specific Enolase, and S-100 Calcium-Binding Protein B 144
Hours After Concussion vs Biomarker Changes After a Friendly Game
PCS <6 d
Biomarker
T-tau, pg/mL
S-100B, μg/L
PCS >6 d
Median (Range)
P
Valuea
Median (Range)
P
Valuea
6.5 (0.93-88.0)
.60
9.5 (4.0-164.8)
.02
0.06 (0.0-0.10)
0.05 (0.03-0.07)
.21
0.06 (0.03-0.23)
.62
8.3 (3.8-12.7)
6.5 (3.2-14.1)
.88
5.4 (3.2-13.8)
.18
After Friendly Game,
Median (Range)
5.8 (0.06-12.45)
Abbreviations: NSE, neuron-specific
enolase; PCS, postconcussive
symptoms; S-100B, S-100
calcium-binding protein B; T-tau, total
tau.
a
NSE, μg/L
after concussion remained significantly elevated in players
with PCS for more than 6 days vs players with PCS for less
than 6 days as well as vs T-tau levels after a friendly game
(Table 2). However, there was no significant difference in
the levels of S-100B and NSE 144 hours after concussion in
players with PCS for less than 6 days and players with persistent PCS or in biomarker changes after a friendly game
(Table 2).
Diagnostic Accuracy of the Biomarkers
The AUC for T-tau 1 hour after concussion vs T-tau after a
friendly game (AUC = 0.80; 95% CI, 0.65-0.94) was greater than
that for S-100B (AUC = 0.67; 95% CI, 0.52-0.83) and NSE
(AUC = 0.55; 95% CI, 0.37-0.70) (Figure 5A). The AUC for Ttau improved to 0.91 (95% CI, 0.81-1.00) when comparing Ttau concentrations 1 hour after concussion in players with PCS
for more than 6 days vs T-tau concentrations after a friendly
game, while the AUCs for S-100B and NSE remained almost unchanged (Figure 5B). Further, the AUC for T-tau 144 hours afE6
Compared with biomarker values
after a friendly game.
ter concussion in players with PCS for more than 6 days vs
T-tau concentrations after a friendly game was also higher
(AUC = 0.76; 95% CI, 0.58-0.94) than that for S-100B and NSE
(Figure 5C).
Discussion
The major finding of this study was that the plasma levels of
T-tau increased in ice hockey players with sports-related
concussion. The highest concentrations of T-tau were measured immediately after the injury, and the levels declined
during the first 12 hours followed by a second peak between
12 and 36 hours. Importantly, T-tau concentrations at 1 hour
after concussion predicted the number of days it took for
the concussion symptoms to resolve and the players to have
safe RTP. The biomarker with greatest diagnostic accuracy
was T-tau, with an AUC of 0.91 when comparing T-tau concentrations in samples collected 1 hour after concussion
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Blood Biomarkers for Brain Injury in Hockey
Original Investigation Research
Figure 5. Diagnostic Accuracy of the Biomarkers
T-tau (AUC = 0.80; 95% CI, 0.65-0.94)
S-100B (AUC = 0.67; 95% CI, 0.52-0.83)
NSE (AUC = 0.55; 95% CI, 0.37-0.70)
T-tau (AUC = 0.91; 95% CI, 0.81-1.00)
S-100B (AUC = 0.68; 95% CI, 0.50-0.87)
NSE (AUC = 0.54; 95% CI, 0.32-0.76)
B
C
100
100
80
80
80
60
40
20
0
Sensitivity, %
100
Sensitivity, %
Sensitivity, %
A
T-tau (AUC = 0.76; 95% CI, 0.58-0.94)
S-100B (AUC = 0.55; 95% CI, 0.33-0.78)
NSE (AUC = 0.65; 95% CI, 0.42-0.89)
60
40
20
60
40
20
0
0
20
40
60
80
100% – Specificity, %
100
0
0
20
40
60
80
100
0
100% – Specificity, %
20
40
60
80
100
100% – Specificity, %
Area under the receiver operating characteristic curve (AUC) for total tau
(T-tau), S-100 calcium-binding protein B (S-100B), and neuron-specific enolase
(NSE) concentrations 1 hour after concussion vs biomarker concentrations after
a friendly game (A) and 1 hour (B) and 144 hours (C) after concussion in players
with persistent postconcussive symptoms for more than 6 days vs biomarker
concentrations after a friendly game.
with T-tau concentrations in samples collected 1 hour after a
friendly game. Further, high T-tau levels at 144 hours after
concussion correlated with persistence of PCS. Although
there was no significant increase in serum levels of S-100B
in postconcussion samples (all times) compared with
preseason samples, S-100B levels at 1 hour after concussion
were signific antly higher compared with preseason
values. There was no significant difference in the levels of
NSE after concussion compared with preseason levels,
and the levels remained unchanged over time following
concussion.
Total tau is one of the most CNS-specific proteins with
high expression in unmyelinated cortical axons.27 To our
knowledge, this is the first study to measure T-tau in serial
plasma samples from professional athletes with sportsrelated concussion. Recently, an ultrasensitive digital
immunoassay for the quantification of tau in serum was
developed, and T-tau levels were measured in patients who
were resuscitated following cardiac arrest. 1 8 In these
patients, the levels of T-tau correlated strongly with clinical
outcome, with the highest levels found in patients with
poor outcome.18 Similar to the patients with cardiac arrest,
using the same method, we also noticed a biphasic release
of T-tau in concussed hockey players. Bimodal kinetics for
biomarkers reflecting tissue damage have also been found
in other disorders, such as acute myocardial infarction in
which a bimodal increase in serum troponin is found.28 The
bimodal increase in plasma tau after mild concussion in
hockey players might in a similar way represent an initial
release of cytosolic tau followed by a later release of
microtubule-bound tau from injured axons.
In the context of TBI, previous studies of T-tau have
been on ventricular CSF, showing increased levels of T-tau
in patients with severe TBI,12,29 and the levels in ventricular
CSF correlate with the severity of injury and clinical
outcome.11,12,29 However, these studies compared the levels
of T-tau in ventricular CSF with levels in controls of lumbar
CSF, which are not comparable owing to the gradient difference between ventricular and lumbar CSF.30 The finding
that T-tau is increased in plasma following sports-related
concussion suggests that this indeed is associated with
axonal injury. In this study, the levels of T-tau normalized
when the players returned to unrestricted competition,
suggesting that T-tau may be a potential biomarker to monitor the course of recovery in athletes with TBI. Further,
the levels of T-tau after concussion correlated with the
duration of PCS, which implies that T-tau levels immediately following concussion may be used to predict the severity of the TBI.
In this study, S-100B levels also increased immediately after a concussion compared with the preseason levels, and the
levels of S-100B at 1 hour after concussion correlated with the
duration of PCS. The S-100B concentration reflects astroglial
injury, and previous studies have shown that serum levels of
S-100B correlate with scores on the Glasgow Coma Scale and
neuroradiologic findings at hospital admission.31 However, a
limitation of S-100B is that it is also expressed in extracerebral cell types, and, as in previous studies,32-36 the levels of
S-100B increased after a friendly game without concussion
compared with baseline.
The levels of NSE remained unchanged over time following concussion as well as compared with the preseason
values. Neuron-specific enolase is a marker of general neuronal injury.37 In the context of TBI, higher levels of NSE
have been reported in CSF from nonsurvivors compared
with survivors following severe TBI, and increased levels
correlate with TBI severity scores in both children and
adults.38-42 The major limitation of NSE in CSF is that it is
highly sensitive to in vitro lysis of erythrocytes from blood
contamination of the samples.43 Further, similar to S-100B,
the levels of NSE increased after a friendly game without
concussion compared with baseline. The findings of this
study argue against NSE as a sensitive marker of neuronal
injury in athletes with sports-related concussion.
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Research Original Investigation
Blood Biomarkers for Brain Injury in Hockey
The main limitations of this study were the relatively small
sample size, which restricts the possibilities of examining biomarker levels in relation to different forms and severities of concussion, and that the biomarkers analyzed might also not be optimal. Total tau is highly CNS specific; however, CSF studies show
that NFL may be a more sensitive marker than T-tau for TBI.13,14
Further, we did not have access to preseason samples for all ice
hockey players, which would have made it easier to evaluate the
longitudinal change in biomarker levels after concussion.
ARTICLE INFORMATION
Accepted for Publication: February 10, 2014.
Published Online: March 13, 2014.
doi:10.1001/jamaneurol.2014.367.
Author Affiliations: Clinical Neurochemistry
Laboratory, Institute of Neuroscience and
Physiology, Sahlgrenska Academy at University of
Gothenburg, Sahlgrenska University Hospital,
Mölndal, Sweden (Shahim, Skillbäck, Blennow,
Zetterberg); Division of Medical Sciences,
Department of Health Sciences, Luleå University of
Technology, Luleå, Sweden (Tegner); Quanterix
Corp, Lexington, Massachusetts (Wilson, Randall);
Department of Surgery, Sahlgrenska University
Hospital, Gothenburg, Sweden (Pazooki); Clinical
Chemistry Laboratory, Sahlgrenska University
Hospital, Mölndal, Sweden (Kallberg); Reta Lila
Weston Laboratories, Department of Molecular
Neuroscience, Institute of Neurology, University
College London, London, England (Zetterberg).
Author Contributions: Drs Shahim and Zetterberg
had full access to all of the data in the study and
take responsibility for the integrity of the data and
the accuracy of the data analysis.
Study concept and design: Shahim, Tegner,
Blennow, Zetterberg.
Acquisition of data: Shahim, Tegner, Wilson,
Randall, Kallberg.
Analysis and interpretation of data: All authors.
Drafting of the manuscript: Shahim, Tegner,
Blennow, Zetterberg.
Critical revision of the manuscript for important
intellectual content: Tegner, Wilson, Randall,
Skillbäck, Pazooki, Kallberg, Blennow, Zetterberg.
Statistical analysis: Shahim, Skillbäck, Blennow,
Zetterberg.
Obtained funding: Tegner, Blennow, Zetterberg.
Administrative, technical, or material support:
Tegner, Wilson, Randall, Skillbäck, Pazooki,
Kallberg, Blennow.
Study supervision: Tegner, Blennow, Zetterberg.
Conflict of Interest Disclosures: Drs Wilson and
Randall, who are employees of Quanterix Corp, and
Drs Zetterberg and Blennow are listed as
coinventors on a US patent application for plasma
tau as a brain injury marker. Dr Blennow has served
on advisory boards for Eli Lilly, Kyowa Kirin Pharma,
Pfizer, and Roche. No other disclosures were
reported.
Funding/Support: This study was supported by
grants from the Swedish Medical Research Council.
Role of the Sponsor: The funder had no role in the
design and conduct of the study; collection,
management, analysis, and interpretation of the
data; preparation, review, or approval of the
manuscript; and decision to submit the manuscript
for publication.
E8
Conclusions
Sports-related concussion in professional ice hockey players
is associated with acute axonal and astroglial injury. Plasma
T-tau, which is a highly CNS-specific protein, is a promising
biomarker to be used both in the diagnosis of concussion and
in decision making as to when an athlete can be declared fit
for RTP.
Additional Contributions: We warmly
acknowledge the participants and the Swedish Ice
Hockey Association. Gisela Malmgård, BSc, Clinical
Neurochemistry Laboratory, Sahlgrenska University
Hospital, Mölndal, Sweden, provided skillful
technical assistance. We thank the medical staff of
the teams for their assistance in diagnosis,
management, and blood sampling of the players:
Bengt Gustafsson, MD, Djurgården Hockey; Torsten
Johansson, MD, PhD, Frölunda HC; Örjan Fröjd, MD;
Henrik Wrethling, MD, Brynäs IF; Karin Rundblad,
MD, Skellefteå AIK; Göran Thoren, MD, Modo
Hockey; Christer Andersson, MD, PhD, Linköpings
HC; Ulf Nordström, MD, Örebro Hockey; Stefan
Serenius, MD, AIK Hockey; Mattias Hell, BSc,
Färjestad BK; Jonas Kalman, MD, HV 71; Stina
Hedin, BSc, Frölunda HC; Patrik Johansson, MD,
Växjö Lakers; and Billy Nilsson, BSc, Rögle BK. None
of these individuals received compensation from
the funding sponsor.
REFERENCES
1. Ellenbogen RG, Berger MS, Batjer HH. The
National Football League and concussion: leading a
culture change in contact sports. World Neurosurg.
2010;74(6):560-565.
2. Hootman JM, Dick R, Agel J. Epidemiology of
collegiate injuries for 15 sports: summary and
recommendations for injury prevention initiatives.
J Athl Train. 2007;42(2):311-319.
3. DeKosky ST, Blennow K, Ikonomovic MD, Gandy
S. Acute and chronic traumatic encephalopathies:
pathogenesis and biomarkers. Nat Rev Neurol.
2013;9(4):192-200.
4. Baugh CM, Stamm JM, Riley DO, et al. Chronic
traumatic encephalopathy: neurodegeneration
following repetitive concussive and subconcussive
brain trauma. Brain Imaging Behav.
2012;6(2):244-254.
5. Alla S, Sullivan SJ, McCrory P, Hale L. Spreading
the word on sports concussion: citation analysis of
summary and agreement, position and consensus
statements on sports concussion. Br J Sports Med.
2011;45(2):132-135.
6. Blennow K, Hardy J, Zetterberg H. The
neuropathology and neurobiology of traumatic
brain injury. Neuron. 2012;76(5):886-899.
7. Hall RC, Hall RC, Chapman MJ. Definition,
diagnosis, and forensic implications of
postconcussional syndrome. Psychosomatics.
2005;46(3):195-202.
8. Sterr A, Herron KA, Hayward C, Montaldi D. Are
mild head injuries as mild as we think?
neurobehavioral concomitants of chronic
post-concussion syndrome. BMC Neurol. 2006;6:7.
9. Hesse C, Rosengren L, Vanmechelen E, et al.
Cerebrospinal fluid markers for Alzheimer’s disease
evaluated after acute ischemic stroke. J Alzheimers
Dis. 2000;2(3-4):199-206.
10. Nylén K, Csajbok LZ, Ost M, et al.
CSF-neurofilament correlates with outcome after
aneurysmal subarachnoid hemorrhage. Neurosci
Lett. 2006;404(1-2):132-136.
11. Ost M, Nylén K, Csajbok L, et al. Initial CSF total
tau correlates with 1-year outcome in patients with
traumatic brain injury. Neurology.
2006;67(9):1600-1604.
12. Franz G, Beer R, Kampfl A, et al. Amyloid beta
1-42 and tau in cerebrospinal fluid after severe
traumatic brain injury. Neurology.
2003;60(9):1457-1461.
13. Zetterberg H, Hietala MA, Jonsson M, et al.
Neurochemical aftermath of amateur boxing. Arch
Neurol. 2006;63(9):1277-1280.
14. Neselius S, Brisby H, Theodorsson A, Blennow
K, Zetterberg H, Marcusson J. CSF-biomarkers in
Olympic boxing: diagnosis and effects of repetitive
head trauma. PLoS One. 2012;7(4):e33606.
15. Zetterberg H, Smith DH, Blennow K.
Biomarkers of mild traumatic brain injury in
cerebrospinal fluid and blood. Nat Rev Neurol.
2013;9(4):201-210.
16. Zetterberg H, Tanriverdi F, Unluhizarci K,
Selcuklu A, Kelestimur F, Blennow K. Sustained
release of neuron-specific enolase to serum in
amateur boxers. Brain Inj. 2009;23(9):723-726.
17. Graham MR, Myers T, Evans P, et al. Direct hits
to the head during amateur boxing is associated
with a rise in serum biomarkers for brain injury. Int J
Immunopathol Pharmacol. 2011;24(1):119-125.
18. Randall J, Mörtberg E, Provuncher GK, et al. Tau
proteins in serum predict neurological outcome
after hypoxic brain injury from cardiac arrest:
results of a pilot study. Resuscitation.
2013;84(3):351-356.
19. Neselius S, Zetterberg H, Blennow K, et al.
Olympic boxing is associated with elevated levels of
the neuronal protein tau in plasma. Brain Inj.
2013;27(4):425-433.
20. Cifu DX, Walker WC, West SL, et al. Hyperbaric
oxygen for blast-related post-concussion
syndrome: 3-month outcomes [published online
November 20, 2013]. Ann Neurol.
doi:10.1002/ana.24067.
21. Barr WB, McCrea M. Sensitivity and specificity
of standardized neurocognitive testing immediately
following sports concussion. J Int Neuropsychol Soc.
2001;7(6):693-702.
22. McCrea M, Guskiewicz KM, Marshall SW, et al.
Acute effects and recovery time following
concussion in collegiate football players: the NCAA
Concussion Study. JAMA. 2003;290(19):25562563.
JAMA Neurology Published online March 13, 2014
Copyright 2014 American Medical Association. All rights reserved.
Downloaded From: http://archneur.jamanetwork.com/ by a GOTEBORGS UNIVERSITET User on 03/14/2014
jamaneurology.com
Blood Biomarkers for Brain Injury in Hockey
23. McCrory P, Johnston K, Meeuwisse W, et al.
Summary and agreement statement of the 2nd
International Conference on Concussion in Sport,
Prague 2004. Br J Sports Med. 2005;39(4):
196-204.
24. McCrory P, Meeuwisse WH, Aubry M, et al.
Consensus statement on concussion in sport: the
4th International Conference on Concussion in
Sport held in Zurich, November 2012. Br J Sports
Med. 2013;47(5):250-258.
25. Rissin DM, Kan CW, Campbell TG, et al.
Single-molecule enzyme-linked immunosorbent
assay detects serum proteins at subfemtomolar
concentrations. Nat Biotechnol. 2010;28(6):
595-599.
26. Giza CC, Kutcher JS, Ashwal S, et al. Summary
of evidence-based guideline update: evaluation and
management of concussion in sports: report of the
Guideline Development Subcommittee of the
American Academy of Neurology. Neurology.
2013;80(24):2250-2257.
27. Trojanowski JQ, Schuck T, Schmidt ML, Lee VM.
Distribution of tau proteins in the normal human
central and peripheral nervous system. J Histochem
Cytochem. 1989;37(2):209-215.
28. Wu AH, Feng YJ, Moore R, et al; American
Association for Clinical Chemistry Subcommittee on
cTnI Standardization. Characterization of cardiac
troponin subunit release into serum after acute
myocardial infarction and comparison of assays for
troponin T and I. Clin Chem. 1998;44(6, pt
1):1198-1208.
29. Zemlan FP, Jauch EC, Mulchahey JJ, et al. C-tau
biomarker of neuronal damage in severe brain
injured patients: association with elevated
Original Investigation Research
intracranial pressure and clinical outcome. Brain
Res. 2002;947(1):131-139.
30. Jeppsson A, Zetterberg H, Blennow K,
Wikkelsø C. Idiopathic normal-pressure
hydrocephalus: pathophysiology and diagnosis by
CSF biomarkers. Neurology. 2013;80(15):13851392.
31. Mondello S, Muller U, Jeromin A, Streeter J,
Hayes RL, Wang KK. Blood-based diagnostics of
traumatic brain injuries. Expert Rev Mol Diagn.
2011;11(1):65-78.
32. Rothoerl RD, Woertgen C. High serum S100B
levels for trauma patients without head injuries.
Neurosurgery. 2001;49(6):1490-1491, author reply
1492-1493.
33. Anderson RE, Hansson LO, Nilsson O,
Dijlai-Merzoug R, Settergren G. High serum S100B
levels for trauma patients without head injuries.
Neurosurgery. 2001;48(6):1255-1258, discussion
1258-1260.
34. Romner B, Ingebrigtsen T. High serum S100B
levels for trauma patients without head injuries.
Neurosurgery. 2001;49(6):1490, author reply
1492-1493.
35. Stålnacke BM, Ohlsson A, Tegner Y, Sojka P.
Serum concentrations of two biochemical markers
of brain tissue damage S-100B and neurone specific
enolase are increased in elite female soccer players
after a competitive game. Br J Sports Med.
2006;40(4):313-316.
36. Stålnacke BM, Tegner Y, Sojka P. Playing ice
hockey and basketball increases serum levels of
S-100B in elite players: a pilot study. Clin J Sport
Med. 2003;13(5):292-302.
jamaneurology.com
37. Scarna H, Delafosse B, Steinberg R, et al.
Neuron-specific enolase as a marker of neuronal
lesions during various comas in man. Neurochem
Int. 1982;4(5):405-411.
38. Böhmer AE, Oses JP, Schmidt AP, et al.
Neuron-specific enolase, S100B, and glial fibrillary
acidic protein levels as outcome predictors in
patients with severe traumatic brain injury.
Neurosurgery. 2011;68(6):1624-1630, discussion
1630-1631.
39. Chiaretti A, Barone G, Riccardi R, et al. NGF,
DCX, and NSE upregulation correlates with severity
and outcome of head trauma in children.
Neurology. 2009;72(7):609-616.
40. Varma S, Janesko KL, Wisniewski SR, et al.
F2-isoprostane and neuron-specific enolase in
cerebrospinal fluid after severe traumatic brain
injury in infants and children. J Neurotrauma.
2003;20(8):781-786.
41. Berger RP, Pierce MC, Wisniewski SR, et al.
Neuron-specific enolase and S100B in
cerebrospinal fluid after severe traumatic brain
injury in infants and children. Pediatrics.
2002;109(2):31.
42. Ross SA, Cunningham RT, Johnston CF,
Rowlands BJ. Neuron-specific enolase as an aid to
outcome prediction in head injury. Br J Neurosurg.
1996;10(5):471-476.
43. Ramont L, Thoannes H, Volondat A, Chastang
F, Millet MC, Maquart FX. Effects of hemolysis and
storage condition on neuron-specific enolase (NSE)
in cerebrospinal fluid and serum: implications in
clinical practice. Clin Chem Lab Med.
2005;43(11):1215-1217.
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