The Use of Blood Biomarkers to Predict Poor Outcome

After Acute Transient Ischemic Attack or Ischemic Stroke

William Whiteley, PhD, MRCP; Joanna Wardlaw, FRCP; Martin Dennis, FRCP; Gordon Lowe, FRCP;
Ann Rumley, PhD; Naveed Sattar, FRCP; Paul Welsh, PhD; Alison Green, PhD;
Mary Andrews, BSc; Peter Sandercock, FRCP

Background and Purpose—The prediction of death or disability (“poor outcome”) after stroke by validated clinical models
might be improved by the addition of blood biomarker measurements. We investigated whether such measurements
improved the classification of patients into 4 categories of predicted risk of poor outcome: very high, intermediate high,
intermediate low, and very low.
Methods—We prospectively recruited symptomatic patients within 24 hours of ischemic cerebrovascular events. We
measured clinical prognostic variables in each patient. We drew blood soon after admission and measured markers of
inflammation, thrombosis, cardiac strain, and cerebral damage. We assessed poor outcome at 3 months with the
modified Rankin Scale and recovery of symptoms at 24 hours. We measured the association between blood marker
levels and poor outcome after adjustment for stroke severity and age with multivariate logistic regression. Where these
associations were statistically significant, we calculated the net reclassification index.
Results—We recruited 270 patients with acute ischemic cerebrovascular events. At 3 months, 112 patients had a poor
outcome. After adjustment for stroke severity and age, only interleukin-6 and N-terminal pro-brain natriuretic peptide
were significantly associated with poor outcome. The addition of either interleukin-6 or N-terminal pro-brain natriuretic
peptide to National Institutes of Health Stroke Scale and age did not improve the prediction of a poor outcome.
Conclusions—Neither interleukin-6 nor N-terminal pro-brain natriuretic peptide had sufficient predictive power to be of
clinical use to predict poor outcome after stroke. The search for better markers to improve the classification of patients across
clinically relevant boundaries of predicted probabilities of outcome events needs to continue. (Stroke. 2012;43:86-91.)
Key Words: biomarkers 䡲 brain infarction 䡲 prognosis

A fter ischemic stroke, the early prediction of death or

disability (“poor outcome”) is of great interest. Statisti-
cal models, constructed with clinical variables such as age or
neurological impairment, make similar predictions of poor
outcome to experienced stroke physicians.1 Biomarkers of the
processes active in ischemic stroke might add predictive
power to these simple statistical models based on bedside
clinical examination.
Many studies have examined the association between
blood marker levels and poor outcome after stroke.2,3 How-
ever, the associations seen in group data, unless very strong,
do not often lead to better predictions of outcome in individ-
uals.4 To be useful, a given marker should at least improve on
the predictions from validated prognostic variables.
We hypothesized that the addition of biomarker levels to
the National Institutes of Health Stroke Scale (NIHSS) and
age5 would improve the prediction of poor outcome (or
recovery at 24 hours) in patients with ongoing symptoms due
to cerebral ischemia who presented to the hospital soon after
the onset. We decided to measure improvement in prediction
with a novel statistic, the net reclassification index, which
gives a clinically meaningful measure of improvement in
prediction.6
We examined biomarkers of pathophysiological processes
that were plausibly associated with poor outcome after stroke
after a systematic review of the available literature.2
Methods
Ethics Statement
The study was approved by the Scotland A Research Ethics
Committee (REC Reference No. 06/MRE00/119). All patients or
their relatives provided written informed consent.

Received July 26, 2011; accepted August 2, 2011.

Ralph L. Sacco, MD, MS, was the Guest Editor for this paper.
From the Division of Clinical Neurosciences (W.W., J.W., M.D., A.G., M.A., P.S.), Western General Hospital, University of Edinburgh, Edinburgh,
UK; the Division of Cardiovascular and Medical Sciences (G.L., A.R., N.S., P.W.), Royal Infirmary, University of Glasgow, Glasgow, UK; and the SFC
Brain Imaging Research Centre (J.W.), SINAPSE Collaboration, University of Edinburgh, Edinburgh, UK.
The online-only Data Supplement is available at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.111.634089/-/DC1.
Correspondence to William Whiteley, PhD, MRCP, Bramwell Dott Building, Department of Clinical Neurosciences, Western General Hospital,
Edinburgh, EH4 2XU, UK. E-mail william.whiteley@ed.ac.uk
© 2011 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.111.634089

Downloaded from http://stroke.ahajournals.org/

86 by guest on May 31, 2016

Cohort Recruitment
Between March 2007 and February 2009 we included consecutive
patients with ischemic stroke or transient ischemic attack who
presented in normal working hours with symptoms of ⬍24 hours’
duration that were still present at the time of initial assessment.
Because of laboratory availability, patients were only eligible if they
could be recruited within normal working hours. All patients were
assessed at the time of presentation when they still had ongoing
symptoms of their acute “brain attack.” A study neurologist assessed
each patient in the acute stage and recorded prognostic factors for
poor outcome after stroke (age, stroke severity, symptoms in the
previous week consistent with infection, medical history, atrial
fibrillation, NIHSS); medications at admission; electrocardiographic
findings; and time when last seen well. We defined prior cognitive
impairment as a history of cognitive problems before the onset of
symptoms that were sufficient to impair activities of daily living,
elicited by the study neurologist.
For each patient, a panel of experts agreed on a final diagnosis of
confirmed ischemic stroke, transient ischemic attack, or not a
vascular event after considering the presentation, CT or MRI brain
imaging, and clinical course blinded to the results of blood marker
levels. We defined an ischemic stroke as a clinically definite stroke
in a patient whose brain imaging showed either positive evidence of
a relevant ischemic lesion or was normal and excluded intracranial
hemorrhage and stroke mimics and where the symptoms lasted for
⬎24 hours. We used a similar definition for transient ischemic
attack, although symptoms had to last for ⬍24 hours.
Biomarker Measurement
We drew blood from each patient before infusion of any
thrombolytic therapy into 2 2.5-mL ethylenediaminetetraacetic acid
tubes and an 8-mL tube containing clot activator and gel for serum
separation. Samples were transferred on water ice and centrifuged at
3000 revolutions per minute for 10 minutes and the supernatant
stored at ⫺80°C.
In serum and plasma blinded to clinical information, clinical and
research laboratories measured as markers of inflammation: adi-
ponectin, C-reactive protein, intracellular adhesion molecule 1,
interleukin-6 (IL-6), IL-10, matrix metalloproteinase 9, tissue necro-
sis factor ␣, von Willebrand Factor, and white cell count; thrombo-
sis: D-dimer, fibrinogen, and tissue-type plasminogen activator;
cardiac strain: N-terminal pro B-type natriuretic peptide (NT pro-
BNP) and troponin T; and neural and glial damage: tau, S100B, and
creatinine, and glucose (see Supplementary Material for detailed
methods; http://stroke.ahajournals.org).
Outcome Measurement
At 24 hours, we determined whether the presenting symptoms had
resolved completely (to differentiate transient ischemic attack from
ischemic stroke). We ascertained vital status by contacting the
patient’s general practitioner at 3 months after onset and sent
surviving patients a short questionnaire based on the modified
Rankin Scale (mRS) by mail. If a patient failed to return an
interpretable questionnaire by mail, we interviewed them in a
structured way by telephone to measure the mRS. We dichotomized
a patient’s outcome into “poor” if he or she was dependent on others
for activities of daily living (mRS scores 3, 4, and 5) or dead and
“good” if he or she was independent in activities of daily living 3
months after stroke onset (mRS 0, 1, and 2). We determined the
cause of death by inspection of the hospital or general practitioner
records.
Statistical Analysis
We examined the association between biomarker levels and delay to
admission with a series of correlation analyses. We made adjustment
for NIHSS at the time of presentation with multiple linear regression
analysis.
We examined the association between clinical variables and blood
markers with poor outcome at 3 months with a series of univariate
logistic regression analyses. We constructed 2 logistic regression
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Whiteley et al A Prospective Cohort Study 87
models to adjust for potential confounders. First, in a simple model,
we adjusted the association between blood markers with poor
outcome for the baseline NIHSS and age. Then in a second series of
more complex models, we adjusted not only for NIHSS, age, and
premorbid disability, but also for the potentially confounding effects
of infection and statins on the associations with inflammatory
markers and for the potentially confounding effects of current or
previous atrial fibrillation, cardiac failure, or previous cardiac
vascular disease on the associations with cardiac markers. We
measured the crude association of blood markers with recovery at 24
hours and adjusted these associations for NIHSS and age.
The Additional Predictive Value of Blood Markers
Over NIHSS and Age
We assessed if the addition of biomarkers significantly associated
with poor outcome made better predictions of poor outcome than a
model built based on NIHSS and age alone.5 We made the most
conservative estimate of the improvement in prediction after the
addition of blood markers by allowing the association of each
clinical covariate with poor outcome to vary. We assessed changes in
goodness of fit (Bayes information criterion), calibration (Hosmer
Lemeshow ␹2), and discrimination (area under receiver operator
characteristic curves) after the addition of biomarkers to the baseline
clinical model. We calculated the net reclassification index6 across
the clinically relevant thresholds of predicted risk: 0.1 (“very low
risk”), 0.5 (“intermediate”), and 0.9 (“very high risk”). We prespeci-
fied thresholds of ⬍10% and ⬎90% for predicted probability of poor
outcome because we believe that one would need to be very certain
of a good or poor outcome before avoiding treatments such as
thrombolysis or selecting patients for palliative care only. The net
reclassification index examines whether the addition of a biomarker
moves those with poor outcome to higher risk categories more often
than lower risk categories and those with a good outcome to lower
risk categories more often than higher risk categories. Because
Bonferroni adjustments for over 5 comparisons are likely to be too
conservative,7 we considered a 2-tailed P⬍0.01 to be statistically
significant. We analyzed the data with Stata 11.
Results
We recruited 270 patients who presented with ongoing
symptoms due to cerebral ischemia. In 40 patients, the
symptoms resolved completely within 24 hours, and in 230,
they were persistent (Figure 1). At 3 months, a mRS score
was available for 268 (99%) patients and vital status for all
patients. At 3 months, 31 (11%) patients had died and 112
(42%) had a poor outcome. The cause of death was the effects
of the original stroke (21), cancer (3), extracranial hemor-
rhage (2), cardiac arrhythmia (1), myocardial infarction (1),
ischemic colitis (1), heart failure (1), and was unavailable in
1. Blood marker data were missing for some patients,
although each patient had blood drawn. Data appeared to be
missing at random. The clinical characteristics of the study
participants are summarized in Table 1 and the levels of
blood markers are shown in an online Data Supplement.
Clinical Features and Outcome
The risk of poor outcome doubled per decade of patient age
(OR, 2.03; 95% CI, 1.58 –2.62) and per 3-U increase in the
NIHSS (OR, 1.98; 95% CI, 1.63–2.39; Table 1). A patient
with poor outcome was significantly (P⬍0.01) more likely to
have atrial fibrillation, prior cognitive impairment, or have
been admitted more quickly to the hospital. Treatment with
statins before stroke was associated (P⫽0.02) with poor
outcome, but not symptoms of infection at admission
88 Stroke January 2012

270 patients with acute symptomatic ischaemic

statin medication before admission and symptoms of infec-
cerebrovascular diseases tion before stroke attenuated this relationship by a small
amount (OR, 1.86; 95% CI, 1.10 –3.18). Adjustment of the
relationship between loge NT pro-BNP with poor outcome for
a prior diagnosis of cardiac failure, atrial fibrillation at the
time of admission and prior cardiac vascular disease, also
weakened the association to a small degree (OR, 2.09; 95%
270 with known vital status at 3 months CI, 1.11–3.93). For other markers, further adjustment made
268 with modified Rankin at 3 months little material difference to the direction, strength, or statisti-
cal significance of the associations.
There were no important differences in the magnitude or
Modified Rankin and baseline measurement of:
statistical significance of the associations between any of the
•Adiponectin n=259
blood markers and poor outcome when the analyses were
•CRP n=258 repeated in the subgroup of 190 patients with positive
•ICAM-1 n=258 imaging findings.
•IL-6 n=259 There was an association of higher levels of NT-pro BNP
•TNFα n=259 with continuing symptoms at 24 hours, which remained after
•IL-10 n=259 adjustment for NIHSS and age (OR, 2.72; 95% CI, 1.25–
•MMP-9 n=259 5.96). The levels of other markers were not significantly
•vWF n=248 associated with clinical outcome at 24 hours (Supplemental
•White cell count n=266 Figure I).
•D-dimer n=258
•Fibrinogen n=259 The Addition of Blood Markers to NIHSS and Age
•tPA n=258 We tested the addition of those markers associated signifi-
•NT pro-BNP n=250 cantly with poor outcome to a baseline model constructed
•Troponin T n=268
with the covariates NIHSS and age (Table 2). The baseline
•Tau n=267
model was well calibrated and showed good discrimination in
•S100B n=255
this cohort.
The addition of NT pro-BNP or IL-6 to the baseline model
•Creatinine n=265
significantly improved the goodness of fit but made little
•Glucose n=259
additional difference to either the discrimination (measured
Figure 1. Flow diagram of study participants and blood marker by area under the receiver operating characteristic curve) or
measurements.
the calibration of the models. Although a few patients moved
across clinically relevant boundaries of predicted probability
(P⫽0.14). Seven patients were treated with intravenous of poor outcome, these numbers were small and not statisti-
recombinant tissue-type plasminogen activator. cally or clinically significant. The full models are summa-
rized in Supplemental Table III.
Blood Markers and Delay to Admission
The median delay to blood draw from symptom onset was 7.1
hours (interquartile range, 3.2–20.1 hours). Although there Discussion
was a significant correlation (P⬍0.01) between increasing Higher levels of NT pro-BNP and IL-6 were strongly asso-
loge delay to blood taking and lower levels of loge D-dimer ciated with poor outcome 3 months after stroke or transient
(r⫽⫺0.21), loge IL-6 (r⫽⫺0.18), and loge tissue-type plas- ischemic attack after adjustment for age and neurological
minogen activator (r⫽⫺0.13), after making adjustment in impairment. Despite this strong association, the addition of
multivariate linear regression for NIHSS at admission, these either NT pro-BNP or IL-6 to NIHSS and age did not improve
associations were no longer statistically significant. the classification of patients to an important degree into “very
high risk” or “very low risk” of poor outcome 3 months after
Blood Markers and Poor Outcome symptom onset. These findings were not affected by delay to
The relationship between blood marker levels and poor admission, statin prescription, or the presence of symptoms of
outcome was approximately log linear for all markers except prior infection, which was a concern in our previous study of
NT pro-BNP and S100B in which a loge transformation was IL-6 and poor outcome.8 It is therefore unlikely that doctors
log linear (Figure 2). Although higher levels of most of the will find predictive instruments that use the measurement of
markers were positively associated with poor outcome, after IL-6 or NT pro-BNP clinically more useful than age and
adjustment for age and the baseline NIHSS, only the associ- NIHSS to predict short-term functional outcome in patients
ations with IL-6 (75th:25th centile OR, 2.05; 95% CI,1.23– with acute stroke. In addition, only NT pro-BNP was asso-
3.41) and NT pro-BNP (OR, 1.26; 95% CI, 1.24 – 4.12) ciated with failure to recover by 24 hours after symptom
reached statistical significance. onset, although there are wide limits of uncertainty about the
Further adjustment of the association (Supplemental Table estimates of other markers, because the number of patients
II) between IL-6 with poor outcome for the prescription of with complete recovery at 24 hours was small.
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 Whiteley et al A Prospective Cohort Study 89

Table 1. The Association Between Baseline Clinical Features and Poor Outcome (Dead or Dependent on Other for Activities of Daily
Living) 3 Months After Presentation With Acute Transient Ischemic Attack or Ischemic Stroke
Baseline Characteristic All (n⫽268) Good Outcome (n⫽156) Poor Outcome (n⫽112) OR (95% CI) P
Male sex, no. (%) 112 (41.8) 84 (53.9) 46 (41.1) 0.60 (0.37– 0.98) 0.04
Age, y, mean (SD) 74.4 (12.5) 70.6 (12.7) 79.6 (10.0) 2.03 (1.58–2.62)* ⬍0.001
Clinical Variables No. (%) No. (%) No. (%)
Recent infection 25 (9.3) 11 (7.1) 14 (12.5) 1.88 (0.82–4.31) 0.14
Prior cardiac vascular disease 65 (24.3) 35 (22.4) 30 (26.8) 1.26 (0.7–2.22) 0.41
Prior peripheral vascular disease 15 (5.6) 9 (5.8) 6 (5.4) 0.93 (0.32–2.68) 0.89
Prior TIA or stroke 73 (27.2) 34 (21.8) 39 (34.8) 1.92 (1.11–3.30) 0.02
Prior heart failure 22 (8.3) 8 (5.2) 22 (8.3) 2.66 (1.08–6.59) 0.03
AF (before onset or in ED) 73 (27.3) 26 (16.8) 47 (41.2) 3.59 (2.04–6.31) ⬍0.001
Diabetes mellitus 34 (12.7) 19 (12.1) 15 (13.4) 1.12 (0.41–2.30) 0.77
Imaging lesion
Cortical infarction 123 (45.9) 57 (36.5) 66 (58.9) ... ...
Lacunar infarction 40 (14.9) 29 (18.5) 11 (9.8) ... ...
Posterior circulation infarction 21 (7.8) 14 (9.0) 7 (6.2) ... ...
⬎1 territory infarction 4 (1.5) 0 4 (3.6) ... ...
No visible lesion 74 (27.6) 54 (34.6) 20 (17.8) ... ...
No scan 4 (1.5) 2 (1.3)‡ 2 (1.7) ... ...
Continuous Variables Median (IQR) Median (IQR) Median (IQR)
Well to admission, hr† 4.0 (1.7–8.5) 4.8 (2.0–11.7) 2.5 (1.3–6.2) 0.96 (0.92–0.99)‡ 0.01
NIHSS 3 (2–9) 3 (1–4) 9 (3–17) 1.25 (1.18–1.34)§ ⬍0.001
Prior cardiac vascular diseases are: a history of angina, myocardial infarction, or cardiac revascularization.
TIA indicates transient ischemic attack; AF, atrial fibrillation; ED, emergency department; IQR, interquartile range; NIHSS, National Institutes of Health Stroke Scale;
OR, odds ratio; CI, confidence interval.
*Per decade of age.
†Time in hours from when the patient was last seen well to their arrival in the emergency department.
‡Per hour increase.
§Per unit increase.

Our finding of the association between NT pro-BNP and
poor outcome after cerebrovascular disease is consistent with
several published studies.9 –11 Plausible explanations for the
association are: (1) NT-pro BNP is a measure of early cardiac
dysfunction after stroke, which is associated with more severe
stroke and worse outcome; (2) NT-pro BNP is associated
with cardioembolic strokes, which tend to have a worse
outcome that other causes of ischemic stroke; or (3) NT
pro-BNP is released from the brain after cerebral ischemia.
However, despite the association of BNP with many potential
causes of poor outcome after stroke, it does not seem to have
a role in clinical prediction of poor outcome.
The significant association of higher levels of adiponectin
with poor outcome after adjustment for potentially confound-
ing variables is also of interest, although this did not reach our
chosen threshold of statistical significance for this study
(P⫽0.02). In previous studies of the association of adiponec-
tin with death after stroke, lower levels of adiponectin were
associated with worse outcome12; our finding suggested that
this may not be the case.
To be useful for predicting outcome in clinical practice,
markers must either outperform established clinical models
when measured alone or improve predictions when added to
these models. The net reclassification index6 is a measure of
the number of patients who are better classified across
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clinically meaningful thresholds after the addition of a new
biomarker. Because there is no widely agreed definition of
clinically important boundaries for the prediction of poor
outcome after stroke, in this study, we chose boundaries after
discussion with experienced stroke physicians. However,
further work is still needed to determine the boundaries that
are most important to patients and less experienced
physicians.
Limitations
This study did not aim to elucidate the causal role of
particular molecules or the underlying physiological path-
ways. There are many plausible hypotheses about how higher
levels of each marker might cause a poor outcome after
cerebral ischemia. However, even in a perfect observational
study, free of the influence of selection or information bias,
causality is not the only factor that might explain the
observed associations. Other explanations include reverse
causality, the choice of confounders used for statistical
adjustment, and imperfect biomarkers.
We recruited patients with a wide range of severity of
baseline neurological impairments and time points of study
entry up to 24 hours after symptom onset. A study of a large
group of patients with a uniform baseline stroke severity or at
a sooner time would have more power to detect important
90 Stroke January 2012

Unadjusted P Adjusted* P
OR (95% CI) OR (95% CI)
Inflammation
Adiponectin 2.34 (1.60 to 3.41) <0.001 1.61 (1.03 to 2.52) 0.04
CRP 1.26 (1.11 to 1.43) 0.001 1.61 (1.03 to 2.52) 0.06
ICAM 1.04 (0.74 to 1.46) 0.83 1.08 (0.70 to 1.66) 0.71
Interleukin-6 4.00 (2.57 to 6.25) <0.001 2.05 (1.23 to 3.41) 0.006
TNF 1.07 (0.84 to 1.36) 0.57 0.97 (0.72 to 1.31) 0.85
Interleukin-10 1.07 (0.99 to 1.15) 0.07 1.03 (0.95 to 1.11) 0.52 Figure 2. Associations between blood
MMP-9 1.11 (0.84 to 1.47) 0.46 1.19 (0.84 to 1.68) 0.32 marker levels and death or disability at 3
vWF 2.17 (1.48 to 3.18) <0.001 1.53 (0.94 to 2.48) 0.09 months after stroke or transient ischemic
attack. *Adjustment made for National
White cells 1.84 (1.29 to 2.63) 0.001 1.59 (1.00 to 2.53) 0.05
Institutes of Health Stroke Scale and age.
Thrombosis The OR is the ratio of odds of poor out-
D-dimer 3.09 (2.09 to 4.58) <0.001 1.55 (0.97 to 2.47) 0.07 come in the 75th to the 25th centile of
Fibrinogen 1.92 (1.01 to 3.68) 0.05 1.29 (0.57 to 2.92) 0.54 plasma or serum marker to allow compar-
ison of the relative strengths of associa-
tPA 1.48 (1.13 to 1.97) 0.005 1.14 (0.82 to 1.58) 0.43 tions between different markers. Probabil-
Cardiac strain ity values are derived from Wald tests and
determine if the reported OR is signifi-
NT pro-BNP 5.23 (3.16 to 8.66) <0.001 2.26 (1.24 to 4.12) 0.008
cantly different from 1. OR ⬎1 indicates
Troponin T 3.86 (2.12 to 7.01) <0.001 1.72 (0.97 to 3.70) 0.17 higher marker levels are associated with
Cerebral damage worse outcome.
Tau 1.00 (0.98 to 1.03) 0.80 0.99 (0.96 to 1.03) 0.73
S100 b 1.26 (0.99 to 1.61) 0.06 1.16 (0.85 to 1.58) 0.35
Other markers
Creatinine 0.94 (0.64 to 1.37) 0.73 0.98 (0.69 to 1.41) 0.92
Glucose 1.20 (1.00 to 1.44) 0.05 1.20 (0.96 to 1.51) 0.11

0.1 1 10
Adjusted Odds ratio*

improvements in prediction with blood markers in patients
presenting at early time points or with particularly severe (or
mild) strokes. However, then the results would be less
generalizable.
We measured the mRS outcome by a postal questionnaire
and telephone interview, similar to large trials of stroke
treatments,13 although we note that there are problems with
most methods of mRS measurement.14 It is possible that with
this method of outcome measurement, our results were
affected by a nondifferential information bias, which would
tend to weaken the observed associations.
Strengths
This study met almost all the methodological criteria for a
reliable study of prognosis.15 We recruited 270 patients, of
whom 112 had a poor outcome at the end of the study.
Because approximately 10 outcome events per covariate has
been suggested as a “rule of thumb” to ensure logistic
regression models have sufficient power, we could examine
approximately 10 covariates.16 Although some patients had
missing blood marker levels, these were random so unlikely
to have led to an important selection bias. Furthermore, the
study was larger than most of the previous studies of blood
biomarkers and stroke outcome. By including patients with
‘clinically probable” as well as patients with “clinically
definite” stroke, we avoided the selection biases associated
with including only patients with imaging positive strokes,
and therefore our cohort is more likely to be representative of
patients with stroke in routine clinical practice.

Table 2. Performance of Models to Predict Poor Outcome in Patients With Acute Symptomatic Transient Ischemic Attack or Stroke
3 Mo
Goodness of Fit
Calibration Reclassification
Likelihood Bayesian Discrimination Hosmer Lemeshow Net
Ratio Statistic Information Statistic Reclassification
(P) Criterion AUROC (95% CI) P (P) Improvement
NIHSS⫹age Reference ⫺1118 0.83 (0.78 – 0.88) Reference 0.91 Reference
NIHSS⫹age⫹IL-6 ⬍0.01 ⫺1120 0.84 (0.79–0.89) 0.10 0.41 ⫹0% (P⫽0.99)
NIHSS⫹age⫹logeNT pro-BNP 0.01 ⫺1120 0.84 (0.79–0.89) 0.05 0.96 ⫹1% (P⫽0.64)
Net reclassification improvement: the difference in correct and clinically useful classifications between models with and without biomarkers at thresholds of
predicted probability of poor outcome of 0.1, 0.5, and 0.9. Positive values indicate better prediction with the addition of a marker; negative values indicate worse
prediction.
AUROC indicates area under the receiver operating characteristic; NIHSS, National Institutes of Health Stroke Scale; IL-6, interleukin-6; NT pro-BNP, N-terminal
pro-brain natriuretic peptide; CI, confidence interval.

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Conclusions
We have confirmed the associations between blood markers
and poor outcome with effects of a similar magnitude to
previous studies. Although these blood markers were there-
fore of potential clinical value, it is sobering to note that none
of the very plausible biomarkers measured in this study added
a clinically useful degree of prediction of poor outcome after
stroke to that provided by the measurement of the simple
clinical variables, the NIHSS and age. Future studies of blood
markers for predicting outcome after stroke will need to
demonstrate that the candidate marker: (1) has a statistically
independent association with outcome; (2) adds statistically
significant predictive value to a clinical model; and (3)
improves the classification of patients across relevant bound-
aries of predicted probabilities of poor outcome that have
clinical use.
Acknowledgments
We are indebted to the Wellcome Trust Clinical Research Facility,
many research fellows, and the patients. The imaging was conducted
in the SFC Brain Imaging Research Centre at the University of
Edinburgh, a SINAPSE center.
Sources of Funding
W.W. was supported the Chief Scientist’s Office (CAF/06/30) and is
now funded by a UK Medical Research Council Clinician Scientist
Fellowship (G0902303). J.W. was supported by the Scottish Funding
Council through the Scottish Imaging Network, a Platform for
Scientific Excellence Collaboration. The study was funded by The
Chief Scientist’s Office and a National Health Service Lothian
endowment, Research and Education in Medical Neurological
Disorders.
Disclosures
None.
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 The Use of Blood Biomarkers to Predict Poor Outcome After Acute Transient Ischemic
Attack or Ischemic Stroke
William Whiteley, Joanna Wardlaw, Martin Dennis, Gordon Lowe, Ann Rumley, Naveed
Sattar, Paul Welsh, Alison Green, Mary Andrews and Peter Sandercock

Stroke. 2012;43:86-91; originally published online October 20, 2011;

doi: 10.1161/STROKEAHA.111.634089
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SUPPLEMENTARY MATERIAL
We list the methods to measure each individual marker below:

Inflammation

• Adiponectin (μg/ml): we measured total plasma adiponectin with a

commercial enzyme-linked immunosorbent assays (ELISA) (R&D Systems).

The inter-assay coefficients of variation for the assay was < 7%.

• C-reactive protein (CRP) (mg/l): we measured plasma CRP with high-

sensitivity immunonephelometry (Prospec, Dade Behring Milton Keynes,

UK) following the manufacturer's reagents and standards. Intra- and inter-

assay coefficients of variation were 4.7% and 8.3%, respectively.

• Intercellular adhesion molecule-1 (ICAM-1) (ng/ml): we measured plasma

ICAM-1 with a commercially available ELISA (R&D Systems, Abingdon,

UK). The inter-assay coefficient of variation was <7%.

• Interleukin-6 (IL-6) (pg/ml): we assayed serum IL-6 with a high sensitivity

ELISA (R & D Systems, Abingdon, UK). Intra- and inter-assay coefficients of

variation were 7.5% and 8.9%, respectively.

• Tumour necrosis factor-alpha (TNF-α ): (pg/ml): we assayed serum TNF- α with

a high-sensitivity ELISA (R&D Systems, Abingdon, UK). Intra-assay and

inter assay coefficients of variation were 8.4% and 12.5%, respectively.

• Interleukin-10 (IL-10) (pg/ml): we measured serum IL10 with an ELISA assay

(R&D Systems, Abingdon, UK). The inter-assay coefficient of variation was

4.5%.
 • Matrix-metalloproteinase-9 (MMP-9) (ng/ml): we measured serum MMP-9 with

a commercially available sandwich ELISA (R&D Systems, Abingdon, UK).

Intra-assay and inter assay coefficients variation were 4.4% and 10.4%,

respectively.

Thombosis

• von Willebrand factor (vWF) (IU/dL): we measured serum vWF antigen with

an ELISA using rabbit antihuman polyclonal antibodies obtained from

DAKO (High Wycombe, UK). Intra and inter assay coefficient of variation

were 3.3% and 4.2%, respectively.

• D-dimer (ng/ml): we measured plasma levels of fibrin D-dimer with a

commercially available ELISA from Biopool AB, Umea Sweden. The intra

and inter assay coefficients of variation were 4.7 and 5.2%, respectively.

• Fibrinogen (g/l): we measured fibrinogen in plasma by imunonephelometry

(Prospec, Dade Behring Milton Keynes, UK) using the manufacturer’s

reagents and standards. Intra- and inter-assay coefficients of variation were

7.5 and 8.9%, respectively.

• Tissue plasminogen activator (t-PA) (ng/mL): we measured plasma levels of

tissue plasminogen activator (t-PA) antigen with commercially available

ELISAs from Biopool AB, Umea Sweden. The intra and inter assay

coefficients of variation were 6.6% and 6.5%, respectively.

Cardiac Strain
 • N-terminal-pro-brain-natriuretic-peptide (NT pro-BNP) (pg/ml): we measured

serum levels of NT pro-BNP using the Elecsys 2010

electrochemiluminescence analyser (Roche Diagnostics, Burgess Hill, UK)

calibrated using the manufacturer’s reagents. Manufacturer’s controls were

used with limits of acceptability defined by the manufacturer. Low control

coefficient of variation was 6.7% and high control coefficient of variation was

4.9%.

• Troponin T (ng/ml): we determined serum troponin T using the Elecsys 2010

electrochemiluminescence analyser (Roche Diagnostics, Burgess Hill, UK)

calibrated using the manufacturer’s reagents. Manufacturer’s control was

used with limits of acceptability defined by the manufacturer. The abnormal

high (detectable) control coefficient of variation was 2.3%.

Cerebral damage

• Tau (pg/ml): we measured tau in serum with a sandwich ELISA using the

Innotest htau antigen (Innogenetics). The coefficient of variation at 479pg/ml

was 5.8%.

S100 B (pg/ml): we measured S100 B in serum. 96-well microtiter plates were coated

with 200 µL of 0.05 M carbonate buffer containing monoclonal anti-S100 B (Affiniti

Research Products, Exeter, UK). The plates were washed with 0.67 M barbitone

buffer containing 5 mM calcium lactate, 0.1% bovine serum albumin, and 0.05%

Tween and then were blocked with 2% bovine serum albumin and washed again.

Two-hundred microliters of diluted serum (1:1) in 0.67 M barbitone buffer

 containing 5 mM calcium lactate was added in duplicate. After incubation and

washing, horseradish-peroxidase-conjugated polyclonal anti-S100 B (Dako,

Copenhagen, Denmark) was used as a detecting antibody. The o-phenylenediamine

color reaction was stopped with 1 M hydrochloric acid, and the absorbances were

read at 492 and 405 nm. The antigen concentration was calculated from an internal

standard curve ranging from 0 to 250 pg/mL. The coefficient of variation at a

concentration of 263pg/ml was 11%.

Supplementary table 1. Distribution of blood markers of inflammation,

thrombosis, cardiac strain and neuronal and glial damage in patients with acute

cerebrovascular disease.
†
Marker All mRS 0-2 mRS 3-6 P
Median (IQR) Median (IQR) Median (IQR)

Inflammation
Adiponectin (µg/ml) 11.4 (6.8-7.9) 9.2 (5.7-15.5) 14.7 (9.0-19.9) <0.001
CRP (mg/l) 4.2 (1.9-8.3) 3.1 (1.5-6.8) 6.2 (2.5-18.3) <0.001
ICAM (ng/ml) 165 (131-215) 163 (131-2.8) 165 (127 to 220) 0.74
Interleukin-6 (pg/ml) 4.5 (2.1-8.2) 2.9 (1.7-5.7) 7.4 (3.9-10.6) <0.001
TNF (pg/ml) 1.4 (1.2-1.7) 1.4 (1.2-1.7) 1.4 (1.3 to 1.8) 0.27
 †
Marker All mRS 0-2 mRS 3-6 P
Median (IQR) Median (IQR) Median (IQR)

Interleukin-10 (pg/ml) 4.4 (3.3-6.6) 4.3 (3.1-6.0) 4.8 (3.5-7.4) 0.06

MMP-9 (ng/ml) 872 (555-1337) 859 (538-1322) 872 (619-1364) 0.45
vWF (IU/dl) 163 (123-218) 147 (113-204) 187 (140-231) <0.001
9
White cells (x10 cell/l) 8.6 (6.7-10.6) 8 (6.3-10.0) 9.1 (7.3-11.5) 0.002
Thrombosis

D-dimer (ng/ml) 228 (109-440) 158 (88-302) 347 (199-770) <0.001

Ln D-dimer (loge unit)* 5.4 (4.7-6.1) 5.1 (4.5 – 5.7) 5.8 (5.3-6.6) <0.001
Fibrinogen (g/l) 4.8 (4.2-5.8) 4.6 (4.0-5.3) 5.2 (4.5-6.0) <0.001

tPA (ng/ml) 11.1 (8.4-14.6) 10.7 (8.4-12.7) 11.6 (8.3-17.2) 0.06

Cardiac strain

NT pro-BNP (pg/ml) 518 (140-1800) 281 (100-860) 1342 (406-3862) <0.001

LnNTproBNP(logeunit)* 6.3 (5.0-7.5) 5.6 (4.6-6.8) 7.2 (6.0-8.3) <0.001

Troponin T (ng/ml) 0.01(0.01-0.01) 0.01(0.01-0.01) 0.01(0.01-0.02) <0.001

Cerebral damage

Tau (pg/ml) 21 (13-49) 23 (12-51) 21 (13-50) 0.83

S100B (pg/ml) 61 (39-109) 59 (37-94) 69 (43-133) 0.08
Ln S100B (loge unit)* 4.2 (3.7-4.7) 4.1 (3.7-4.6) 4.3 (3.8-5.0) 0.03
Other markers

Creatinine (µmol/l) 86 (68 to 104) 83 (68-102) 86 (69-109) 0.19

Glucose (mmol/l) 5.8 (5.2 to 6.8) 5.6 (5.1-6.7) 6.0 (5.4-7.3) 0.02
* variables loge transformed to give a log-linear relationship with outcome in logistic regression; †Mann Whitney U

test. mRS: modified Rankin scale

Supplementary table 2 Associations between marker levels with death or disability at 3

months.
Marker (units) OR, with further P- value
*
adjustment
(95% CI)
Inflammation
Adiponectin (µg/ml) 1.79 (1.12 to 2.85) 0.02
CRP (mg/l) 1.11 (0.98 to 1.26) 0.10
ICAM (ng/ml) 1.06 (0.68 to 1.65) 0.79
Interleukin-6 (pg/ml) 1.86 (1.10 to 3.18) 0.02
TNF (pg/ml) 0.86 (0.62 to 1.18) 0.34
 Interleukin-10 (pg/ml) 1.03 (0.95 to 1.11) 0.52
MMP-9 (ng/ml) 1.21 (0.85 to 1.73) 0.28
vWF (IU/dl) 1.45 (0.87 to 2.42) 0.15
9
White cells (x10 cell/l) 1.59 (0.98 to 2.55) 0.06
Thrombosis

D-dimer (loge unit) 1.52 (0.95 to 2.45) 0.08

Fibrinogen (g/l) 1.33 (0.88 to 2.01) 0.17

tPA (ng/ml) 1.18 (0.85 to 1.65) 0.32

Cardiac strain

NT pro-BNP (loge unit) 2.09 (1.11 to 3.93) 0.02

Troponin T >0.01ng/ml 1.84 (0.86 to 3.94) 0.12
Cerebral damage

Tau (pg/ml) 0.99 (0.95 to 1.03) 0.63

S100 b (loge unit) 1.16 (0.84 to 1.61) 0.36
Other markers

Creatinine (µmol/) 0.94 (0.64 to 1.37) 0.73

Glucose (mmol/l) 1.19 (0.94 to 1.50) 0.14

Adjustment made for *NIHSS and age, independence of activities of daily living and for

markers of inflammation: prior infection and prescription of statins; and for cardiac strain

markers: cardiac failure, AF and prior cardiac vascular disease. The OR is the ratio of odds of

poor outcome in the 75th to the 25th centile of plasma or serum marker. P-values are derived

from Wald tests and determine if the reported OR is significantly different from 1.
Supplementary table 3 Predictive logistic regression models to predict poor outcome

at 3 months after presenting with acute symptomatic TIA or stroke

Variables NIHSS + age Loge NT pro-BNP IL-6 +

+ NIHSS + age NIHSS + age

Age* 1.07 (1.03 to 1.10) 1.04 (1.01 to 1.08) 1.06 (1.03 to 1.09)
†
NT pro-BNP 2.26 (1.24 to 4.12)
†
IL-6 1.99 (1.19 to 3.33)
‡
NIHSS 1.23 (1.15 to 1.32) 1.20 (1.12 to 1.29) 1.20 (1.12 to 1.29)

Constant -6.37 -6.90 -6.31

*per year; †75th to 25 centile; ‡per unit increase;

Odds ratios and 95% confidence intervals for variables in each model. Odds

ratios for each variable from each logistic regression model (i.e. eβ).

The constant terms come from the underlying logistic regression model of the

form: logit (poor outcome)= βa.a + βb.b + constant

 Unadjusted P Adjusted P
OR (95% CI) OR (95% CI)
Inflammation
Adiponectin 1.17 (0.73 to 1.91) <0.51 0.82 (0.48 to 1.43) 0.49
CRP 1.62 (1.04 to 2.53) 0.03 1.25 (0.86 to 1.82) 0.23
ICAM 1.04 (0.74 to 1.46) 0.83 1.08 (0.70 to 1.66) 0.71
Interleukin-6 2.62 (1.34 to 5.10) 0.005 1.29 (0.63 to 2.62) 0.50
TNF 1.26 (0.87 to 1.82) 0.22 1.17 (0.83 to 1.64) 0.91
Interleukin-10 0.98 (0.94 to 1.03) 0.46 0.90 (0.81 to 1.01) 0.07
MMP-9 0.92 (0.63 to 1.34) 0.65 0.86 (0.56 to 1.33) 0.49
vWF 1.64 (0.97 to 2.78) 0.07 1.14 (0.66 to 1.97) 0.64
White cells 1.48 (0.84 to 2.61) 0.18 1.90 (1.11 to 3.23) 0.02
Thrombosis
D-dimer 2.59 (1.52 to 4.39 <0.001 1.60 (0.87 to 2.96) 0.12
Fibrinogen 1.51 (0.94 to 2.42) 0.09 1.10 (0.66 to 1.82) 0.72

tPA 1.32 (0.87 to 1.98) 0.18 1.00 (0.62 to 1.61) 0.99

Cardiac strain
NT pro-BNP 4.49 (2.38 to 8.47) <0.001 2.72(1.25 to 5.96) 0.01
Troponin T 3.86 (2.12 to 7.01) <0.001 1.72 (0.97 to 3.70) 0.17
Cerebral damage
Tau 1.01 (0.96 to 1.05) 0.74 1.00 (0.95 to 1.05) 0.99
S100 b 1.40 (0.40 to 5.3) 0.62 0.94 (0.22 to 4.04) 0.94
Other markers
Creatinine 1.32 (0.84 to 1.09) 0.23 1.12 (0.96 to 1.07) 0.66
Glucose 1.18 (0.86 to 1.62) 0.31 1.06 (0.79 to 1.43) 0.70

0.1 1 10
Adjusted odds ratio

Supplementary figure 1 Associations between blood marker levels and failure to recover by
24 hours after symptom onset after stroke or TIA

*Adjustment made for NIHSS and age. The OR is the ratio of odds of poor outcome in the 75th to the 25th
centile of plasma or serum marker to allow comparison of the relative strengths of associations between
different markers. P-values are derived from Wald tests and determine if the reported OR is
significantly different from 1. OR>1 indicate higher marker levels are associated with worse outcome