Brain & Development 26 (2004) 398402

www.elsevier.com/locate/braindev

Original article

Neuron-specific enolase as a marker of the severity and outcome

of hypoxic ischemic encephalopathy
ner, O
zer Pala
Coskun Celtik*, Betul Acunas, Naci O
Department of Pediatrics, Trakya University Faculty of Medicine, Edirne, Turkey
Received 7 April 2003; received in revised form 24 December 2003; accepted 24 December 2003

Abstract
The aim of this study was to evaluate serum concentrations of neuron-specific enolase (NSE) as a marker of the severity of hypoxic
ischemic encephalopathy (HIE) and to elucidate the relation among the concentrations of NSE, grade of HIE and short-term outcome. Fortythree asphyxiated full-term newborn infants who developed symptoms and signs of HIE (Group 1) and 29 full-term newborn infants with
meconium-stained amniotic fluid but with normal physical examination (Group 2) were studied with serial neurological examination, Denver
developmental screening test (DDST), electroencephalogram and computerized cerebral tomography (CT) for neurological follow-up. Thirty
healthy infants were selected as the control group. In the patient groups, two blood samples were taken to measure NSE levels, one between 4
and 48 h and the other 5 7 days after birth. Serum NSE levels were significantly higher in infants with HIE compared to those infants in
Group 2 and control group. The mean serum concentrations of the second samples decreased in all groups studied but they were significantly
higher in Group 1 compared to those in Group 2. Serum NSE concentrations of initial samples were significantly higher in patients with stage
III HIE than in those with stages II and I. The sensitivity and specificity values of serum NSE as a predictor of HIE of moderate or severe
degree (cut-off value 40.0 mg/l) were 79 and 70%, respectively, and as a predictor of poor outcome (cut-off value 45.4 mg/l) were calculated
as 84 and 70%, respectively. The predictive capacity of serum NSE concentrations for poor outcome seems to be better than predicting HIE
of moderate or severe degree. However, earlier and/or CSF samples may be required to establish serum NSE as an early marker for the
application of neuroprotective strategies.
q 2004 Elsevier B.V. All rights reserved.
Keywords: Neuron-specific enolase; Perinatal asphyxia; Hypoxic ischemic encephalopathy; Short-term outcome

1. Introduction
Despite advances in medical and technological possibilities, perinatal asphyxia is still a matter of concern due to its
considerably high rate of mortality and morbidity [1,2].
Hypoxic ischemic encephalopathy (HIE) after perinatal
asphyxia is a condition in which cerebrospinal fluid (CSF)
and/or serum concentrations of brain-specific biochemical
markers may be elevated [3 7]. Recent development of
neuroprotective strategies emphasize the need for early and
objective indicators for both diagnosis and outcome of HIE
and studies about biochemical markers may aid neuroprotective studies [8].
niversitesi Tp Fakultesi,
* Corresponding author. Address: Trakya U
Cocuk Saglg ve Hastalklar Anabilim Dal, 22030 Edirne, Turkey. Tel.:
90-284-235-7641; fax: 90-284-235-2338.
E-mail address: cceltik2001@yahoo.com (C. C
eltik).
0387-7604/$ - see front matter q 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.braindev.2003.12.007

Of those brain-specific proteins, the brain-derived

creatinine kinase (CK-BB), the highly soluble glial protein
S-100, glial fibrillary acidic protein (GFAp), neurofilament
protein (NFp) and the neuron-specific enolase (NSE) have
been found to be released in high concentrations into the
CSF of asphyxiated infants and correlated significantly with
other indicators of long-term prognosis and neurological
impairment at 1 year of age or death [4]. NSE, a dimeric
glycolytic enzyme, containing two gamma subunits,
originates predominantly from the cytoplasm of neurons
and neuroendocrine cells. It is soluble and stable in
biological fluids and its determination is not affected by
hyperbilirubinemia or lipemia. Furthermore it does not elicit
immunological cross-reactivity with non-neuronal enolase
[3 7]. Serum levels of NSE increase in neuroendocrine cell
tumours, medulloblastoma, retinoblastoma, neuroblastoma,
seminoma, small cell cancer of lung, brain hypoxia after

C. Celtik et al. / Brain & Development 26 (2004) 398402

myocardial infarction, cell damage associated with central

and peripheral nervous system as stroke, subarachnoidal
haemorrhage, traumatic brain damage, Guillian Barre
syndrome, bacterial meningitis and encephalitis. NSE has
been established both as a useful and reliable marker of
neuronal damage and prognosis in these various neurological disorders [9 15]. On the other hand there are few
studies about its value in the diagnosis and prognosis of HIE
following perinatal asphyxia most of which have used CSF
values of NSE as an indicator of neurological impairment
[3 7]. However, it is important to have a sufficiently
sensitive marker for brain damage that can be determined in
blood instead of CSF, because blood samples can be taken
more easily and frequently and more independently of
raised intracranial pressure than CSF samples and can be
performed in all conditions even in haemorrhagic diathesis
associated with severe asphyxia.
The aim of this study was to evaluate serum concentrations of NSE as a marker of the severity of HIE and to
elucidate the relation between the concentrations of NSE
and grade of HIE in relation to short-term outcome.

2. Material and methods

The patient group comprised of 43 asphyxiated full-term
newborn infants who developed symptoms and signs of HIE
according to Sarnat and Sarnat [2] and treated at Neonatal
Intensive Care Unit of Trakya University Hospital
(Group 1). Infants were designated as having asphyxia if
they fulfilled the following criteria.
(1) Intrapartum distress indicated by the cardiotocograph
pattern (late decelerations, absence of variability,
persistent bradycardia, etc.) and/or abnormal blood
flow pattern (loss or reversal of end-diastolic velocity)
and/or early passage of thick meconium,
(2) Requirement for resuscitation with positive pressure
ventilation and laryngeal intubation,
(3) Low Apgar score (1st min # 3, 5th min , 6) or
umbilical arterial/first postnatal (pH , 7.1).
Twenty-nine full-term infants with meconium-stained
amniotic fluid but with normal physical examination comprised Group 2. Thirty healthy newborn infants who were
born after spontaneous vaginal delivery with normal physical
findings were included as the control group. Infants with
severe congenital abnormalities, profound anaemia, and history of multiple pregnancies were excluded from the study.
Gender gestational age, mode of delivery, birth weight,
weight for gestational age, Apgar scores at 1st and 5th min,
if present the stage of HIE [mid HIE (stage I), moderate HIE
(stage II) and severe HIE (stage III)] [2], neurological
disabilities of survivors were recorded.
In Group 1, neurological examination was performed
everyday during the hospitalization period, and 1 week, 3, 6

399

and 12 months after discharge. Electroencephalographic

(EEG) and computerized tomography examinations (CT) of
the brain were performed at 1 month and 12 months of age
in all patients in Group 1. During the first year of life, every
month, Denver Developmental Screening Test II (DDST)
adapted for Turkish children [16] was performed in Group 1.
Physical examination and DDST were performed in all
patients in Group 2 and control group at 1 month of age and
repeated every 3 months. Patients were examined by the
same neonatologist (BA) and DDST was performed by
paediatric neurodevelopmental specialist.
Poor outcome was overall determined after 1 year of
delivery. It was defined as death due to HIE or presence of
abnormal neurological findings, that is, abnormalities in
muscular tonus, uncoordinated or absent sucking and
swallowing, the presence of major disabilities such as
spasticity, seizure, hearing loss or neurodevelopmental
delay supported by either persistently abnormal EEG
findings or DDST and/or CT results. EEG findings such as
voltage suppression, burst suppression, isoelectric tracing,
diffuse paroxysmal activity or localized periodic epileptiform discharge, the existence of diffuse hypoxic hypodense
areas and related findings on the brain CT imaging were
accepted as signs of poor prognosis. Besides a child was
identified as having poor outcome by DDST when delays in
development significantly challenged the child in two or
more of the following four developmental areas: personal
social, fine motor adaptive, language, and gross motor.
Normal or good outcome was defined as the absence of
those disabilities mentioned above.
Informed parental consent was obtained for all infants
before the collection of blood samples.
In the patient groups, two blood samples were taken to
measure NSE levels, one between 4 and 48 h (except in one
case at 52 h of age) and the other, between 5 and 7 days after
birth. The blood samples were taken at similar time periods
from all groups. Only one sample has been taken from those
in the control group and those who were lost during followup or died. All blood samples were immediately frozen
(2 70 8C) and haemolysed samples were discarded to
prevent false high value of NSE.
Serum NSE concentrations were measured using the
NSE radioimmunossay kit (Pharmacia AB, Uppsala,
Sweden) and according to the manufacturers instructions.
The method measures concentrations in the range of
2 200 mg/l; the detection limit is less than 2 mg/l.
2.1. Statistics
All values were presented as mean and SD. The
distribution of serum NSE concentrations were assessed
by Kolmogorov Smirnov and Shapiro Wilk tests. x 2 -test
was used to compare gender, place of birth (inborn/outborn), mode or delivery. For comparing three groups in
terms of birth weight and gestational ages which were
parametric data, analysis of variance (ANOVA) test, and to

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eltik et al. / Brain & Development 26 (2004) 398402

compare Apgar scores, NSE1 and NSE2 levels which were

non-parametric data, Kruskal Wallis test was used. Kruskal Wallis test was also used to compare HIE subgroups in
terms of serum NSE concentrations. The significance of
differences between groups was analysed by post hoc Tukey
HSD test and Mann Whitney U-test. The specificity,
sensitivity, positive and negative predictive values of NSE
levels in determining diagnosis and prognosis of HIE were
obtained using optimal cut-off levels and were calculated on
the material used in this study. Receiver operating
characteristics (ROC) curves were assessed using the
areas under the curves as indexes of performance. For
statistical analysis Minitab Release 13 (license number: wep
1331.00197) for Windows was used. P , 0:05 was
considered as statistically significant.

3. Results
The demographic data of all groups are shown in Table 1.
There were no differences between patient and control
groups in terms of gestational age, birth weight, gender and
mode of delivery. More patients in Group 1 were outborn
ones (P , 0:05; Group 1 vs Group 2 and control group).
The first and fifth minute Apgar scores of patients in Group 1
were significantly lower than those in Group 2 and the
control group (P , 0:001; Group 1 vs Group 2 and control
group). Eighteen infants in Group 1 (14%) had meconiumstained amniotic fluid. Fourteen patients in the HIE group
were classified as in stage I, 19 as in stage II, 10 as in
stage III. While no morbidity and mortality had been
observed in Group 2 and the control group, 8 patients died
(2 in stage II HIE group and 6 in stage III HIE group; for this
reason second blood samples of these patients are lacking)
and 11 patients were observed to have poor outcome in
Group 1 after 1 year of follow-up. The mean first blood
sampling time was 20 ^ 14 (4 52) h for Group 1; 14 ^ 12
(4 48) h for Group 2; 15 ^ 7 (4 26) h for control group.
The mean second blood sampling time was 6.5 ^ 0.7 and
6.6 ^ 0.9 d for Groups 1 and 2, respectively. There were no

Table 2
Serum concentrations of NSE at different sampling times (NSE1, NSE2) in
the two patient groups and the control group and according to the stage of
HIE
Group

NSE1 (mg/l)

NSE2 (mg/l)

Group 1, stage I HIE n 14

Group 1, stage II HIE n 19
Group 1, stage III HIE n 10
Group 2 n 18
Control group n 30

65.3 ^ 32.4a
64.6 ^ 32.9a
115.7 ^ 60.9a
42.0 ^ 24.00c
21.0 ^ 5.3c

34.6 ^ 13.9b
42.0 ^ 32.7b
70.8 ^ 36.9b
22.1 ^ 8.0

a
b
c

Pstage I vs III and Pstage II vs III , 0:05:

Pstage I vs III and Pstage I II , 0:05:
Pgroup1 vs 2 , 0:05; Pgroup1 vs control , 0:001:

significant differences among the groups in terms of first and

second blood sampling time. The second blood samples
were not taken from the controls.
There were significant differences between Group 1 and
Group 2 and controls in terms of serum NSE concentrations of the first blood samples (Pgroup 1 2 , 0:05;
Pgroup 1 control , 0:001).
Serum concentrations of NSE at different sampling times
(NSE1, NSE2) in the two patient groups and the control
group and according to the stage of HIE are shown in
Table 2. No significant difference was detectable in serum
NSE levels between Group 2 and the Control group. The
mean serum concentrations of the second samples decreased
in all groups studied, however, they were significantly
higher in Group 1 compared with Group 2 P , 0:05:
Serum NSE concentrations of the first blood samples
were significantly higher in patients with stage III HIE than
those with stage I and II HIE (Pstage I III and Pstage II III ,
0:05; Pstage I II , 0:05). Fig. 1 demonstrates the NSE
concentrations in relation to outcome (poor and normal).
Initial NSE levels were evaluated by ROC-curve analysis
in which sensitivity and specificity were calculated for
different cut-off values to distinguish the patients who

Table 1
Characteristics of the study groups and controls

Birth weight (g)

Gestational age (week)
Gender (M/F)
Inborns/outbornsa
Mode of delivery, CS/NSVDb
Apgar score (1 min)c
Apgar score (5 min)c
a
b
c

Group 1
n 43

Group 2
n 29

Control
n 30

3119 ^ 604
39.8 ^ 1.6
28/15
6/37
17/26
1.9 ^ 1.3
4.0 ^ 1.0

3218 ^ 559
39.6 ^ 0.9
16/13
27/2
15/14
6.9 ^ 1.7
8.7 ^ 0.8

3436 ^ 437
39.4 ^ 0.9
12/18
19/11
/30
8.2 ^ 0.4
9.3 ^ 0.5

P , 0:05; Group 1 vs Group 2 and control group.

CS, caesarean section; NSVD, normal spontaneous vaginal delivery.
P , 0:001; Group 1 vs Group 2 and control group.

Fig. 1. Serum concentrations of NSE

poor).

12

in relation to outcome (normal,

C. Celtik et al. / Brain & Development 26 (2004) 398402

401

positive and negative predictive values of serum NSE as a

predictor of HIE of moderate or severe degree were 79, 70,
51 and 89%, respectively, and as a predictor of poor
outcome were calculated as 84, 70, 39 and 95%,
respectively.

4. Discussion

Fig. 2. ROC curves for initial NSE (cut-off point 40.0) as a marker for
distinguishing infants with no or mild HIE from infants with moderate or
severe HIE.

developed HIE of moderate or severe degree from those

with no or mild HIE and those who had poor outcome from
those with good outcome. The cut-off values that maximized the sum of sensitivity plus specificity were 40.0 mg/l
for predictor of HIE and 45.4 mg/l for poor outcome (Figs. 2
and 3). By using these values the sensitivity, specificity,

Fig. 3. ROC curves for initial NSE (cut-off point 45.4) as marker for
distinguishing infants with poor outcome from infants with normal
outcome.

Clinical trials of neuronal rescue therapies need to select

those infants who are most likely to benefit from treatment
and to avoid exposing infants who have a good outcome to
potentially toxic therapies. Therefore, it is very important to
find an early and reliable indicator of the severity degree of
HIE and poor outcome to initiate or end neuroprotective
strategies. In this present study, we have demonstrated that
serum NSE concentrations of infants with stage III HIE
were significantly higher than those in stage I and II HIE.
Several studies measuring NSE and other biochemical
indices in serum and/or CSF of infants with HIE following
perinatal asphyxia showed almost similar results [3 6].
Garcia et al. [3] and Blennow et al. [4] reported higher CSF
NSE levels in newborn babies with grade-II and III HIE than
the ones with grade-I HIE and normal control subjects.
Thornberg et al. [5] found high NSE levels in two cases of
grade-II HIE patients and in all grade-III HIE patients. They
reported that all of these babies had severe neurological
impairment and NSE levels were correlated with cerebral
function. However, Nagdyman et al. [7] reported no
significant difference in serum NSE levels between infants
with no or mild and moderate or severe HIE, 2 and 6 h after
birth but there was a significant difference at 12 and 24 h
age. Yet, in the same study the sensitivity, specificity,
positive and negative predictive values of NSE in predicting
moderate or severe HIE (cut-off value 46 mg/l) were found
to be 83, 65, 42 and 93%, respectively [7], which is not so
different from our results (cut-off value 40.0 mg/l; 79, 70,
51, and 89%, respectively). Besides Nagdyman et al. [7] had
also reported sensitivity, specificity, positive and negative
predictive values at cut-off value 4.6 mg/l for protein S100
as 71, 86, 63, and 90%, and at cut-off value 17.0 U/L for
CK-BB as 86, 77, 55, and 94%, respectively [7], which are
almost similar to those belonging to NSE values obtained in
their study. There were also no clear differences between
ROC curves of different biochemical markers for perinatal
asphyxia.
Optimal blood sampling time for biochemical markers to
indicate neuronal damage is controversial. In different
studies associated with neuronal damage due to perinatal
aspyxia, sampling time shows great variance; in most of
these studies including ours, blood and/or CSF samples
were obtained mostly after 6 h of age; Garcia et al. [3]
obtained samples at 12 and 72 h of age; Thonberg et al. [5]:
between 2 and 64 h after birth, Nagdyman et al. [7] at 2, 6,
12 and 24 h after birth. In our study, the reason for this
delay was the high number of outborn patients especially in

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eltik et al. / Brain & Development 26 (2004) 398402

the HIE group. Studies in perinatal animals suggest quick

cellular destruction after hypoxia and a steady decrease in
energy substrates of brain in 48 h [17,18]. Therefore
choosing sampling time as the first 48 h after birth seemed
suitable initially. On the other hand, animal studies indicate
that greater neuroprotection is obtained if hypothermia,
which is the most promising neuroprotective strategy
nowadays, is started soon after the hypoxic insult, possibly
within 6 h of birth [8]. Unfortunately, we could not obtain
blood samples earlier, which shadows the results of this
study. Earlier samples and/or NSE in cerebrospinal fluid can
be more favourable in predicting the severity of HIE and
poor outcome.
The preceding studies associated with NSE about
perinatal asphyxia mostly focused on early diagnosis of
HIE. However, there are few studies investigating the
relation of NSE and HIE outcome in the neonatal period
[3,6]. In this study, both the evaluation of NSE values for
early diagnosis of HIE and the role of NSE for prediction of
HIE outcome were researched and the sensitivity and
specificity of NSE in predicting poor outcome (at cut-off
value 45.4 mg/l: sensitivity 84%; specificity 70%; positive
predictive value 39%; negative predictive value 95%) were
found to be better than predictive values for severity degree
of HIE. Garcia et al. [3] noted that specificity and sensitivity
of initial NSE levels in the assessment of poor outcome, if
cut-off value was 25 ng/ml, were 86 and 90%, respectively.
On the other hand, Verdu Perez et al. [6] noted higher ratios
of sensitivity and specificity for blood NSE as a predictor of
poor outcome (100 and 78%, respectively).
In this study, although sensitivity and specificity rates of
NSE were found to be moderately high for the evaluation of
diagnosis and poor outcome of HIE, positive and negative
predictive values were relatively low. This can be a more
favourable condition if you consider the necessity of having
high sensitivity rate in order to identify those infants with
HIE due to perinatal asphyxia and initiate neuroprotective
therapies. On the other hand in order to prevent unnecessary
intensive follow-up, prediction of HIE outcome, specificity
and negative predictive values that differentiate healthy
infants becomes more important than sensitivity and
positive predictive values. From this point of view,
determination of serum NSE values may be considered as
a reliable assay for detecting and following up neonates with
HIE due to perinatal asphyxia.
In conclusion, the results of this study demonstrate that
the predictive capacity of serum NSE concentrations for
poor outcome seems to be better than predicting HIE of
moderate or severe degree. However, earlier and/or CSF
samples are required to establish serum NSE as an early
predictor for the application of neuroprotective strategies.

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