Early Human Development 105 (2017) 63–67

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Early Human Development

journal homepage: www.elsevier.com/locate/earlhumdev

The potentials and limitations of neuro-biomarkers as predictors of

outcome in neonates with birth asphyxia☆
Angela Satriano b, Francesca Pluchinotta b, Francesca Gazzolo a, Laura Serpero a, Diego Gazzolo a,⁎
a
Dept. of Maternal, Fetal and Neonatal Medicine, “C. Arrigo” Children's Hospital Alessandria, Italy
b
Dept. of Cardiology and Laboratory Research, S. Donato Milanese University Hospital, Milan, Italy

a r t i c l e i n f o a b s t r a c t

Keywords: Perinatal asphyxia and its complication, hypoxic-ischemic encephalopathy, are still among the major causes of
S100B perinatal mortality and morbidity. Despite accurate standard postnatal monitoring procedures, the post-insult
Perinatal asphyxia period is crucial because at a time when radiologic pictures are still silent, brain damage may already be at a sub-
Brain damage clinical stage. Against this background, the measurement of quantitative parameters, such as constituents of ner-
Newborn vous tissue, that are able to detect subclinical lesions at a stage when routine brain monitoring procedures are still
HIE
silent, could be particularly useful.
Therefore, in the present review we report the potentials and limitations of biomarkers in predicting outcome in
neonates complicated by perinatal asphyxia.
© 2016 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
About 0.1–0.4% of full-term newborns (FTN) can be affected by peri-
natal asphyxia (PA) and its dramatic complication, hypoxic-ischemic
encephalopathy (HIE) [1]. PA-HIE accounts for 15–20% of perinatal mor-
tality and an additional 25% develop childhood disabilities. While mild
HIE is known to be at relatively low risk for motor or cognitive deficits,
in severe HIE the common clinical repertoires are cerebral palsy (CP)
and intellectual disabilities. Moreover, in moderate HIE motor deficits,
memory impairment and visual motor or visual perceptive dysfunction,
increased hyperactivity and delayed school readiness can occur [2,3].
HIE pathogenesis is fairly complex and still not fully understood. The
developing fetal brain is highly reliant on a constant sustained blood
flow and, during hypoxic-ischemic insult (H-I), different intracellular
mechanisms are activated, which in turn lead to cell damage [4].
Abbreviations: FTN, full-term newborns; PA, perinatal asphyxia; HIE, hypoxic-
ischemic encephalopathy; CP, cerebral palsy; H-I, hypoxic-ischemic insult; CNS, central
nervous system; BM, biomarkers; GFAP, glial fibrillary acidic protein; NSE, neuron
specific enolase; AM, adrenomedullin; FDA, food and drug administration; EMA,
european medicine agency; MRI, magnetic resonance imaging; CSF, cerebrospinal fluid;
ICH, cerebral hemorrhage; PPV, positive predictive value; NPV, negative predictive
value; PLR, positive likelihood ratio; NLR, negative likelihood ratio; BBB, brain blood
barrier; HT, hypothermia.
☆ Under the Auspices of the Italian Society of Neonatology and The I.O. PhD International
Research Program
⁎ Corresponding author at: Dept. of Maternal, Fetal and Neonatal Medicine, “C. Arrigo”
Children's Hospital, Spalto Marengo 46, I-15100 Alessandria, Italy.
E-mail addresses: angela.satriano@gmail.com (A. Satriano),
francesca.pluchinotta@gmail.com (F. Pluchinotta), francesca.gazzolo@libero.it
(F. Gazzolo), tinal1@hotmail.it (L. Serpero), dgazzolo@hotmail.com (D. Gazzolo).
The intensity, severity and timing of asphyxia, as well as peculiar is-
chemic susceptibility and the degree of maturity of the brain, define the
dimension and grade of severity of the consequent damage [4]. There
are several circumstances in which the developing brain is especially
vulnerable to ischemia [5], and late preterm infants born between 34
and 36 weeks of gestation are known to be at highest risk of permanent
neurological sequelae [6]. The reason for this is that at this stage central
nervous system (CNS) growth is at its peak in terms of brain weight,
volume, structure and function [6].
The pathophysiological steps leading to CNS damage need to be up-
dated: there is evidence that, in addition to the bi-phasic post-H-I insult,
the so-called third phase, which may last for weeks, months and even
years, has to be included [7,8]. The mechanisms of this persisting dam-
age, which are still a matter of debate, involve gliosis, persistent inflam-
matory receptor activation and epigenetic changes.
The combined effects of the above pathophysiological phases act by
disrupting essential components of the cell, leading ultimately to death
or apoptosis [7,8] and to a significant increase in peculiar CNS
neurobiomarkers (BM) in biological fluids such as brain constituents
(activin A; glial fibrillary acidic protein, GFAP; neuron specific enolase,
NSE; S100B) and vasoactive agents (adrenomedullin, AM).
In the present review we report the potentials and limitations of bio-
markers in predicting outcome in neonates complicated by PA.
2. Biomarkers: where are we?
In recent years there has been increased interest in the usefulness of
BMs for the early diagnosis of CNS damage, as well as for the monitoring

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64 A. Satriano et al. / Early Human Development 105 (2017) 63–67
and evaluation of the extent of the lesion. The Food and Drug Adminis-
tration (FDA) and the European Medicines Agency (EMA) have been
shown to support research on BMs [9,10]. The main points of interest re-
gard the specificity of BMs for determining the extent of CNS damage
and the timing of the insult. Briefly, a BM may enable clinicians to: (i)
screen infants for brain injury with high sensitivity and specificity, (ii)
monitor the progression of disease through longitudinal monitoring,
thanks to their short half-life, (iii) correlate the findings of BMs with
those of validated standard procedures able to diagnose brain injury
and the extent of lesions, such as ultrasound and magnetic resonance
imaging (MRI). In addition to these criteria, the new generation of
BMs in perinatal medicine have the following properties: (i) they have
been well studied in the pediatric/neonatal population; (ii) they can
be measured using kits that are commercially available worldwide, are
simple to use and provide measurements with good reproducibility;
(iii) reference ranges are available for pediatric/neonatal populations;
(iv) they can be assessed in different biological fluids such as urine,
blood, cerebrospinal fluid (CSF), amniotic fluid, saliva and milk, possibly
reducing perinatal stress related to testing.
3. Biomarkers
3.1. Activin A
Activin A is a member of the transforming growth factor-β super-
family, which in the CNS regulates neurons differentiation and prolifer-
ation [10,11]. The neuroprotective action of activin A is exerted via the
activation of different pathways: in animal models and cell cultures
activin A has a beneficial role in neuronal recovery, supporting the sur-
vival of neurogenic cell lines and retinal neurons and offering protection
against neurotoxic damage [11]. In rats, activin A enhances neuron sur-
vival, rescues against neurotoxic damage and decreases H-I injury [11].
3.1.1. Potentials
Activin A has been measured in milk and in CSF, cord and peripheral
blood and urine of PA-HIE FTN [10]. In CSF, activin A concentrations
were higher in PA FTN who developed severe HIE than in those who
did not or in controls. An activin A cut-off of 1.3 pg/L as an early diagnos-
tic test for HIE [12] had a sensitivity and specificity of 100% with a pos-
itive predictive value (PPV) of 100% and a negative predictive value
(NPV) of 0%. Notwithstanding these promising results, the assessment
of activin A in CSF has been gradually abandoned due to the invasive
sample collection procedure and stress for infants. In blood, elevated
longitudinal activin A levels in PA-FTN correlated with impaired fetal
circulation and hypoxia [13]. As an HIE diagnostic test an activin A
cut-off of 0.66 ng/L achieved a sensitivity of 93.33%, a specificity of
96.63%, with positive and negative likelihood ratios (PLR, NLR) of
27.69 and 0.069, respectively. In both blood and CSF, activin A increased
in PA-HIE before the appearance of related clinical and laboratory signs
[14]. More recently, activin A levels measured in urine were significantly
higher in PA newborns with moderate or severe HIE than in those with
absent or mild HIE. An activin A cut-off N 0.08 ng/L at first void had a sen-
sitivity of 83.3% and a specificity of 100% for predicting the development
of moderate or severe HIE [15].
These observations suggest that activin A could provide additional
information to physicians on the occurrence of perinatal brain injuries
at a stage when diagnostic procedures have a limited benefit.
3.1.2. Limitations
To date, few data are available regarding the correlation of activin A
levels with the extent of brain lesions, assessed using standard tech-
niques such as ultrasound and MRI, and with short-term outcomes. Ref-
erence curves in the neonatal/pediatric period are requested due to the
trophic role of activin in CNS development [9–11,16].
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3.2. GFAP
A monomeric protein of the astroglial skeleton with a molecular
mass of 40–53 kDa located in the white and gray matter [9,10] where
it can be released rapidly out of damaged areas and up-regulated
through astrogliosis. Changes in blood concentrations of GFAP have
also been shown to reflect damage to the blood-brain barrier (BBB).
3.2.1. Potentials
GFAP has been investigated mainly in conventional biological fluids
(i.e. CSF, blood) of PA infants. In CSF, GFAP was assayed between 12 and
48 h after birth of PA-HIE FTN. GFAP levels increased up to 5-fold in accor-
dance with HIE severity [17]. Indeed, GFAP measured in the first 4 days of
life was significantly increased and correlated with other indicators of
long-term prognosis and with neurological impairment at 1 year of age,
or death before that time [17,18]. The PPV of a GFAP measurement higher
than the 98th percentile of normal infants was 69%, while a GFAP level
below this limit invariably predicted a good outcome [18].
In blood, increased GFAP levels are closely correlated with the sever-
ity of HIE. At a GFAP cut-off of 0.08 pg/mL, the PPV value was 100% and
NPV 0% for predicting infants with abnormal outcomes at 15–18 months
of age [19]. Reports also showed that GFAP at a cut-off value of
0.07 ng/mL, had a sensitivity and specificity for HIE of 77% and 78%, re-
spectively, and correlated with MRI patterns [20].
3.2.2. Limitations
Few data are currently available regarding reference curves for GFAP
in different biological fluids. This is of relevance, bearing in mind that
GFAP is reasonably involved in CNS development and is therefore gesta-
tional age-dependent. Notably, data on GFAP levels in unconventional
biological fluids are still lacking, and no conclusive results have been re-
ported regarding correlations between GFAP and cerebral ultrasound
and MRI patterns.
3.3. NSE
An enzyme that catalyzes the conversion of 2-phospho-D-glycerate
to phosphoenolpyruvate in a glycolytic pathway. It has a molecular
weight of 78 kDa and a half-life of 24 h. In the CNS it is characterized
by its stable presence in mature sensory and endocrine neurons, sug-
gesting that a unique system of intracellular energy metabolism may
be shared by neurons and paraneurons [9,10]. Immunohistochemistry
shows that NSE can be traced in the cerebral circulation after experi-
mentally induced cerebral lesions, and plasma concentrations correlat-
ed with events of neuronal NSE loss in focally ischemic neurons [9,10].
NSE is also present in erythrocytes, platelets, plasmatic cells, lym-
phocytes, capillary walls, and myoepithelial cells, which explains its
physiologically low concentrations in blood. NSE is secreted after cell in-
jury into the extracellular fluids, CSF and blood, where it represents an
attempt to maintain homeostasis [21].
3.3.1. Potentials
NSE has been investigated mainly in conventional biological fluids
(i.e. CSF, blood) of PA infants and is thought to be released into the
blood as a consequence of damage to BBB, or brain injury due to tissue
ischemia or edema [22]. In blood, higher NSE levels were observed in
PA-HIE FTN than in controls. At a cut-off value of N 40.0 mg/L NSE
achieved a sensitivity and specificity as a predictor of moderate/severe
HIE of 79% and 70% respectively, and a PPV and NPV of 51% and 89%, re-
spectively. Moreover, NSE as a predictor of poor outcome at a cut-off
value N 45.4 mg/L had a sensitivity and specificity of 84% and 70%, and
a PPV and NPV of 39% and 95%, respectively [23]. Conversely, Nagdyman
et al. found no differences in NSE concentrations in FTN with no or mild
HIE and those with moderate or severe HIE [24]. Roka et al. [25] mea-
sured NSE blood concentrations in PA infants subjected to whole-body
hypothermia (HT) and found that NSE levels were highly elevated
 A. Satriano et al. / Early Human Development 105 (2017) 63–67
following PA, decreased during HT and were strongly correlated with
the neuro-developmental outcome at 2 years of age.
3.3.2. Limitations
Although NSE has been suggested as a promising biomarker in peri-
natal medicine, few reports are currently available time. Among the lim-
itations to its use, the fact that it is unstable in all conditions in which
hemolysis occurs (NSE is expressed on red blood cell membranes) is
its main weakness [9,10]. To our knowledge, investigations providing
correlations between NSE, the extent of brain lesion (MRI) and short/
long-term follow-up are lacking. Furthermore, no reference curves are
yet available for the perinatal period or in different biological fluids.
3.4. S100B
An acidic calcium-binding protein of the EF-hand family, highly spe-
cific for CNS, where it appears to be most abundant in glial cells,
Schwann cells and neurons [9,10]. The protein has a half-life of 1-h
and is eliminated mainly by the kidneys (98%) [26]. As an extracellular
factor, S100B engages receptors for advanced glycation end products
in a variety of cell types with different outcomes (i.e. beneficial or detri-
mental, pro-proliferative or pro-differentiative) depending on the con-
centration of the protein, the cell type and the microenvironment. It
has been implicated in several cellular processes, particularly those in-
volving calcium homeostasis and signal transduction, in which S100B
seems to play a dual role in the regulation of cell function, being benefi-
cial to cells at low doses but detrimental at high doses [27,28].
As a rather small protein with a molecular weight of 21 kDa in its bi-
ologically active homodimeric form, S100B passes the BBB, and is thus
detectable as a brain-derived protein in peripheral blood [29].
3.4.1. Potentials
S100B has been widely studied in different biological fluids such as
CSF, amniotic fluid, blood, urine, saliva and milk [30–39]. More recently,
in high-risk pregnancies the protein has been detected in fetal-maternal
bloodstreams [40,41]. In perinatal medicine, S100B in CSF has been used
to monitor newborns affected by PA and post-cerebral hemorrhage ven-
tricular dilatation (ICH) [18,19,42]. S100B correlated with the extent of
brain lesions, with long-term prognosis, and with neurological impair-
ment at 1 year of age or death before that time. S100B in blood was mea-
sured hypothesizing that during active brain injury the protein may be
released from the damaged tissue and part of it could spread into the
systemic circulation [43], also as a result of hemodynamic rearrange-
ment of the BBB. Increased S100B concentrations were detected 48–
72 h before any clinical, laboratory, or ultrasound signs of cerebral hem-
orrhage in FT-HIE newborns [44]. Nagdyman et al. [24] reported that
S100B in cord blood was already significantly higher in PA-HIE new-
borns. At 2-h from birth and a protein cut-off of 8.5 μg/L, the PPV of
S100B for HIE was 71%, the NPV 90%, sensitivity was 71%, and specificity
90% [28]. S100B also correlated with abnormal cerebral hemodynamic
patterns and with the extent of ICH in FT-HIE newborns [44].
In urine, longitudinal S100B measurement has also been investigated
in PA-HIE FTN [45–47]. An early increase in S100B levels was a predictor
of HIE and subsequent adverse neurological outcomes [45]. An S100B cut-
off of 0.28 μg/L at first urination had a sensitivity of 100% and a specificity
of 87.3% for predicting adverse 1-year neurological follow-up. The sensi-
tivity and specificity of measurements obtained from 12 to 72 h after
birth were up to 100% and 98.2%, respectively. PPV and NPV were 46.2
and 100%, respectively. Moreover, elevated S100B levels have been
shown to represent a marker of brain damage and HIE. An S100B cut-off
of 0.41 μg/L at first urination had a sensitivity of 91.3% and a specificity
of 94.6% for predicting the development of HIE. The sensitivity and spec-
ificity of measurements obtained from 4 to 72 h after birth were up to
100% and 98.8%, respectively. PPV and NPV were 80.8 and 97.8 respective-
ly [46]. More recently, S100B has also been detected in saliva which is the
preferred fluid option in terms of timing and longitudinal collection, as
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65
well as because of the relatively low stress of the sampling procedure.
S100B saliva levels increased in FT PA-HIE and correlated with the occur-
rence of abnormal MRI patterns and with adverse 1-year neurological
outcome. At a cut-off value N3.25 MoM, S100B achieved a sensitivity
and a specificity of 100% as a single marker for predicting the occurrence
of abnormal neurological outcome [48].
Finally, Roka et al. measured S100B blood concentrations in PA in-
fants subjected to HT. They found that S100B levels were significantly
elevated following PA, decreased during HT and were closely correlated
with the neuro-developmental outcome at 2 years of age [25].
3.4.2. Limitations
Of the BMs considered here, S100B is one of the most thoroughly stud-
ied. Reference curves in different biological fluids (i.e. amniotic, cord and
peripheral blood, urine, saliva) have been provided [31–38]. Results show
that the protein is gestational age-, gender- and mode of birth delivery-
dependent. However, before including S100B in daily practice there are
some points that need further investigation. In particular: i) correlation
with cerebral ultrasound and MRI patterns in preterm and term infants
and, ii) correlation with long term neurological outcome (≥2 years).
3.5. AM
It is a C-amidated peptide belonging to the calcitonin gene-related
peptide family, first isolated from human pheochromocytoma and in-
volved in the response to hypoxia and inflammation, which are associ-
ated with neovascularization [9,10]. In the CNS, AM is expressed mainly
in neurons and in the endothelium, where it has been proven to have a
role in vasodilatation, although other actions have also been reported,
including neuromodulation and inhibition of apoptosis [49]. In the rat
model, AM is detectable in the hypothalamus and in the neuronal nuclei
of the caudate-putamen area, one of the brain areas most sensitive to
H\\I damage, suggesting its involvement in response to hypoxia [50–
52]. The findings in animal models support the notion that AM acts as
a neuro-protective and vascular-neuro-regenerative factor [53].
3.5.1. Potentials
AM has been shown to participate in the cascade of events promot-
ing fetal/neonatal cardiovascular adaptation [9,10]. Its assessment in
CSF and in cord and peripheral blood provides useful information in
cases complicated by PA and HIE [9,10]. Early elevated AM blood con-
centrations have been found to predict cases at risk for HIE and ICH
[54]. Of note, in newborns complicated by congenital heart diseases el-
evated longitudinal AM assessment in the peri-operative period corre-
lated with the occurrence of adverse neurological outcome at 1-year
follow-up [55]. AM as an early diagnostic test for adverse neurological
outcome at a cut-off of 17.4 ng/L achieved a sensitivity of 100% and a
specificity of 73.0% with PLR and NLR of 3.7 and 0.0, respectively.
3.5.2. Limitations
Further investigation is needed to determine correlations between
AM, the extent of a brain lesion, cerebral ultrasound and MRI patterns
and short/long-term follow-up. Reference curves in neonatal/pediatric
patients are still lacking, although at this stage there is no evidence
that AM is gestational age- and/or gender-dependent.
4. Conclusions
According to FDA and EMA requirements further progress is needed
before BMs or combinations of BMs can become daily practice in NICUs.
However, as for adult and pediatric diseases we are not so far [56–58].
There are still a few issues that need to be addressed: the most regard
the different techniques for assessing BMs (i.e. ELISA, LIASION, HPLC)
in terms of reproducibility, sensitivity, specificity and optimal timing
for obtaining results. This latter issue is crucial for the selection of pa-
tients suitable for treatment and to employ neuro-protective strategies
66 A. Satriano et al. / Early Human Development 105 (2017) 63–67
Table 1
Optimality items for a biomarker (BM) according to the following international criteria: marker of brain damage and of degree and extension of the lesion; possibility of longitudinal mon-
itoring; studied in pediatric/neonatal populations and provided of reference curves; measurable by commercial kits; measurable in different biological fluids.
BM Brain Damage Degree of injury Lesion Extension Longitudinal Monitoring
Activin A Y Y Y Y
GFAP Y Y Y Y
NSE Y Y Y Y
S100B Y Y Y Y
AM Y Y Y Y
Abbreviations: GFAP, glial fibrillary acid protein; NSE, neuron specific enolase; AM, Adrenomedullin; Y, yes; N, no; CS, cerebrospinal; A, amniotic; C, cord blood; P, peripheral blood; U,
urine; S, saliva.
at the appropriate time. Another issue regards the role of BMs as an
early diagnostic tool and as a reliable indicator of the effectiveness of
neuro-protective strategies. In this respect, the results of multicenter in-
vestigations in wider populations are eagerly awaited.
In summary, as shown in Table 1, S100B protein appears the best op-
tion among BMs, while other BMs such as activin A and AM need further
investigation in saliva. Studies on GFAP and NSE in unconventional bio-
logical fluids are still needed.
5. Future prospects
Recent advances in laboratory technology should lead to the inclu-
sion of novel BMs in the panel of analytes potentially useful for the mon-
itoring and early detection of damage to the CNS. The goal of the BM
panel is to provide, in addition to standard monitoring procedures, use-
ful information on infants complicated by PA-HIE regarding: i) their
suitability for HT treatment; ii) the effectiveness of therapeutic strate-
gies, iii) the risk of short/long-term CNS damage. Although the advent
of therapeutic HT in newborns with moderate HIE has considerably im-
proved the prognosis, results from cases of severe HIE are still scarce
[59]. It is also important that HT is a time-sensitive intervention with a
very narrow therapeutic window and must be performed within 6 h
of delivery to be effective [59]. Thus future challenges for neonatolo-
gists, biochemists, and neuroscientists will involve the adoption of
other therapeutic strategies in addition to HT in order to improve the
short/long-term prognosis of PA-HIE FT infants. In this regard, BM will
constitute, together with MRI, the best tools for longitudinal monitoring
of treatment effectiveness. It is therefore to be hoped that BMs will yield
further details that will help physicians: i) to predict the progression of
disease, ii) to identify injured brain regions, and iii) to determine the
timing of the injury, thus shedding light on the probable pathophysiol-
ogy and the most effective therapy. Preliminary data on the assessment
of BMs in maternal blood in high-risk pregnancies will thus be the key
targets of investigation for early determination of the timing of insults
and treatments.
Among recently available biomarkers for brain injury identified in
newborns ubiquitin carboxy-terminal hydrolase L-1 protein and oxida-
tive stress BMs also deserve further investigation [60].
Last but not least, metabolomics appears to be a highly promising tech-
nique and encouraging preliminary results have been reported in high-
risk newborns [61]. However, further investigations are needed in wider
populations before this approach can be included in clinical guidelines.
Conflict of interest statement
None of the authors has any financial or personal relationships with
other individuals or organizations that could inappropriately influence
their work.
Acknowledgement
This work is part of the I.O. PhD International Program under the
auspices of the Italian Society of Neonatology and was partially supported
by grants to DG from “I Colori della Vita Foundation”, Italy (2015/001).
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Pediatric Population Available kit Reference curve Biological fluid
N Y N CS, A, C, P, U
N Y N CS, C, P
N Y N CS, C, P
Y Y Y CS, A, C, P, U, S
N Y N CS, A, C, P
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