Identifying neurologic biomarkers is important for improving outcomes after perinatal brain injury. Validated biomarkers can optimize neurotherapeutic trials by more accurately risk stratifying patients at entry and serving as surrogate outcomes. If they enter clinical care, biomarkers can be used to help guide therapy, assess treatment effects, and offer prognostic information for families. Substances that can be measured in accessible biological fluids offer advantages over current tools for assessing the degree of brain injury in this population. Most centers rely on clinical examination or Sarnat staging30
to identify patients needing therapy. However, clinical examination changes over time, is subjective, and often confounded by neuroactive medications and interference from medical support devices. The initial clinical examination was recently shown to be a less reliable predictor of outcome in infants treated with hypothermia.31
Electroencephalographic data, although a useful prognostic tool,32,33
requires equipment and interpretive expertise not available at many centers. The emerging simplified amplitude integrated electroencephalogram tool was recently demonstrated by us and others to have limited early predictive value in infants treated with hypothermia.34,35
Even though specificity and positive predictive value improve after the first 48 hours of life, this improvement coincides with a decrease in sensitivity (approximately 50% at rewarming) for identifying infants with adverse outcome.35
MRI, although predictive of outcome,28,36–39
has limited value in the first 24 hours of life8,9
and is restricted by the impracticability of transporting a critically ill neonate for imaging. Thus, there is a need for markers of brain injury that can be reliably measured and objectively interpreted. Data from this study suggest that elevated serum S100B and NSE are associated with brain injury evident during the neonatal period by either MRI findings or persistent neurologic abnormalities at 2 weeks. This association with outcome is independent of baseline characteristics and clinical assessment of encephalopathy at presentation.
S100B is a calcium-binding protein concentrated in astroglial cells of the central nervous system. Although its biological function has not been fully elucidated, S100B is neurotrophic at physiologic concentrations and neurotoxic at high concentrations.10,40
Supraphysiological concentrations have been described in the setting of acute traumatic brain injury12–14
S100B is useful as a biomarker because of its relative ease and reproducibility of measurement, ready detectability in a variety of biologic fluids (eg, serum), and potential for longitudinal monitoring given its short (1 hour) half-life.40
Its utility has been evaluated in small studies of infants with neonatal encephalopathy, with conflicting results.17,18,20,23,24
This variability in findings might be attributable to the limited sample sizes, rarity of moderate or severe encephalopathy and adverse outcome in the study groups, and variable measurement times that characterize the majority of previous studies. Our data demonstrate that S100B (and NSE) levels change significantly over the first 24 hours of life, making group comparisons of measurements taken at nonuniform time points across subjects difficult to interpret. Specifying a narrow time window for comparable measurements is critical when evaluating the predictive ability of dynamic protein levels. Our results provide evidence that S100B reflects the extent of brain injury in infants with encephalopathy and provides predictive values at specific time points relevant to clinical practice.
NSE is a glycolytic enzyme concentrated in the cytoplasm of neurons and released in the setting of cell death.11
It has been studied extensively in pediatric traumatic brain injury,12–14
but only a few studies have been conducted in infants with encephalopathy. Several investigations have demonstrated an association between cerebrospinal fluid (CSF) NSE and severity of encephalopathy after asphyxia, as well as prediction of later neurodevelopmental outcome.19,22,41
The utility of a CSF biomarker is limited, however, because CSF it is not routinely sampled in infants presenting with encephalopathy, and indeed sampling is often contraindicated in the most critically ill patients. NSE measured from serum has also been evaluated, but with conflicting results.
We have evaluated S100B and NSE as potential biomarkers of brain injury in encephalopathic newborns undergoing hypothermia. Because hypothermia has emerged as the only proven effective neuroprotective therapy for newborns with encephalopathy,3–6
investigations into future therapeutic interventions in this population will be performed as adjuvants to cooling (ie, “hypothermia plus” trials). Thus, it is essential that studies evaluating potential biomarkers be performed in the setting of hypothermia. Validated biomarkers can then be used to identify patients who may benefit from adjuvant therapies or possible variations in hypothermia protocols.
Some limitations of this study should be acknowledged. To establish whether these potential biomarkers offer additional information over baseline characteristics or clinical variables assessable at the bedside, potential confounding variables were entered into the multiple regression models. The covariables explored might not represent all important variables that could be related to outcome. Likewise, fully accounting for all factors that could possibly have different effects on biomarker levels among subjects was not possible. It is possible that elevated S100B levels in infants with poor outcome are related to overwhelming systemic disease rather than specific for brain injury, given that infants with encephalopathy often present with multiorgan disease and that extraneural sources of S100B (including muscle, kidney, heart, and adipose tissue) have been reported.42
In addition, serum protein levels might not truly reflect extent of injury to the central nervous system, because these measurements can be affected by the integrity of the blood-brain barrier and other factors that control release of proteins into the periphery in the setting of brain injury. Finally, technical variables, such as time to processing, effect of hemolysis, and effect of temperature, may affect the reproducibility and reliability of results. In light of these limitations, and the fact that that neither biomarker demonstrated perfect discriminatory ability, identifying a single ideal biomarker may remain an elusive goal. Some investigators have proposed that examination of a biomarker panel for consistency of results indicating brain injury might improve the prognostic abilities of proteins considered individually.43
This study did not include a control group of healthy, non-encephalopathic infants. However, because the primary goal was to evaluate whether these biomarkers could identify infants with significant brain injury among at-risk infants, including a healthy control group was not necessary for our study aims. Serum levels in our overall population were higher than normative values reported from healthy newborns on the first day of life in previous studies (ie, S100B, 0.68 ± 0.29 ng/mL44
; NSE, 21 ± 5.3 ng/mL21
Specifying definitions of neuroradiographic and clinical adverse outcomes was an important aspect of this study. These short-term outcomes are surrogates for later significant neurologic disability. Although there is clear evidence that severe MRI-detected abnormalities are highly predictive of later neurologic deficits,28,36–39
the predictive value may be subject to variability due to differences in imaging protocols (ie, timing of acquisition, image parameters and sequences used), as well as in interrater reliability of the neuroradiologists’ interpretation. Significant neurologic deficit 2 weeks after insult was evaluated as a second clinical endpoint. The definition used in this study describes a severe degree of neurologic devastation, a phenotype highly associated with disability later in life.29,45
There was clear overlap between these outcomes, because all patients with significant neurologic deficit had injury detected on MRI. Likewise, all patients with severe MRI-detected injury had abnormalities on neurologic examination, although not all met the defined criteria for significant deficit. The findings for both neuroradiographic and clinical outcomes indicate that S100B and NSE are associated with significant brain injury. Clearly, some patients that did not meet these criteria may develop neurologic impairment later in life. Further work is needed, and is ongoing as a secondary aim of this study, to correlate S100B and NSE levels with long-term outcome.