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Technical Briefs |
Departments of
1
Pediatrics and
2
Obstetrics and Gynecology, Giannina Gaslini Children’s University Hospital, I-16147 Genoa, Italy
3
Institute of Anatomy, Catholic University, Largo Francesco Vito, 1 I-00168 Rome, Italy
aauthor for correspondence: fax 39-630154813, e-mail fabrizio.michetti{at}rm.unicatt.it
Intraventricular hemorrhage (IVH) is the most common form of
cerebral hemorrhage in preterm infants, affecting 
15–20%
(1). Despite accurate postnatal monitoring, IVH is difficult
to diagnose during the first 72 h after birth because at this
stage, clinical symptoms and radiologic assessment of brain damage may
still be silent (2). The availability in these patients of
quantitative indicators suggesting subclinical lesions at a
time when the monitoring indicators are unable to detect bleeding is
therefore important. Furthermore, quantification of the extent of the
hemorrhaged lesion could permit the prevention and/or treatment of
clinical neurologic damage.
S100B is an acidic calcium-binding protein found in the nervous system, where it is concentrated in glial cells (3). The presence of increased S100B in the blood and cerebrospinal fluid (CSF) is a consolidated marker of brain damage in adults and children (4)(5)(6)(7)(8) and has been proposed recently for use in preterm newborns developing IVH (9). We have detected the S100B protein in the urine of preterm newborns under normal conditions and established reference values for the protein at the first urination (10). The present work, extending from the earlier study, investigates urine S100B in preterm newborns developing IVH to offer an indicator for the early detection of IVH.
We performed a case-control study on 18 preterm newborns (29–35 weeks of gestation) with IVH in whom urine S100B was measured at first urination (time 0) and at 24 (time 1), 48 (time 2), and 72 h of age (time 3). IVH was diagnosed at 72 h after birth by ultrasound scanning (11): eight infants were classified as grade II (IVH without ventricular dilatation); eight as grade III (IVH with ventricular dilatation); and two as grade IV (IVH with parenchymal lesion). No other ultrasound abnormalities were described at different monitoring time points. Standard cerebral ultrasound was performed by a real-time ultrasound machine (Acuson 128SP5) at the same time points as urine sampling.
After admission to the neonatal intensive care unit, all newborns routinely underwent clinical assessment (measurement of red blood cell count, venous blood pH, ion concentrations, plasma glucose concentrations, arterial blood pressure), cerebral ultrasound, and neurologic examination according to Prechtl (12). We classified neurologic conditions using a qualitative approach, assigning each infant to one of the diagnostic groups: "normal", "suspect", or "abnormal". An infant was considered abnormal when one or more of the following neurologic syndromes were present: hyper- or hypokinesia, hyper- or hypotonia, hemisyndrome, apathy syndrome, and hyperexcitability syndrome. An infant was classified as suspect if only isolated symptoms were present, but no defined syndromes. The same examiner, who did not know the sonogram outcomes of the infants, tested all infants after IVH was diagnosed. After IVH was diagnosed, clinical and laboratory results and cerebral ultrasound scans recorded at the predetermined monitoring time points were reevaluated and compared with those obtained from a control group. The latter consisted of 18 age-matched preterm infants who were admitted consecutively to the neonatal intensive care unit and did not develop IVH (1 control infant for each IVH infant). Exclusion criteria were as follows: multiple pregnancies, maternal hypertension, diabetes and infections, clinical evidence of chorioamnionitis, premature rupture of membranes (>24 h), fetal malformations, and chromosomal abnormalities.
Emergency cesarean section was performed in 8 of 18 cases in the IVH group and in 9 of 18 cases in the control group; indications included placental abruption and nonreassuring fetal status as defined by the American College of Obstetricians and Gynecologists (bradycardia, late decelerations in heart rate, severe and repetitive variable decelerations in heart rate, reduced beat-to-beat variability). Corticosteroid therapy was administered [Bentelan (betamethasone), 12 mg/24 h for 2 days intramuscularly; Glaxo Wellcome] in 10 of 18 cases in the IVH group and in 11 of 18 cases in the control group. Nine of 19 women who delivered vaginally received tocolytic therapy [Miolene (ritodrine), 30–50 mg/h intravenously; Lusofarmaco]. In 19 infants (10 in the IVH group and 9 in the control group), the birth weight was <10th percentile for gestational age: none of the newborns was delivered by forceps.
The Ethics Committee of the Giannina Gaslini Children’s Hospital, Genoa University, approved the study protocol and the parents of the infants examined gave informed consent.
At the indicated time points, urine samples were immediately
centrifuged at 900g for 10 min, and the supernatants were
stored at -70 °C before measurement. We measured the S100B protein
concentration in all samples, using a commercially available
immunoluminometric assay (Lia-mat Sangtec 100; AB Sangtec Medical).
According to the manufacturer’s instructions, this assay distinguishes
between the A1 and B subunits of the S100 protein and measures the B
subunit as defined by the three monoclonal antibodies SMST 12, SMSK 25,
and SMSK 28. The B subunit of the S100 protein is known to be
predominant (80–96%) in the human brain
(13)(14). Each measurement was performed in
duplicate according to the manufacturer’s recommendations, and the
means were reported. As indicated by the manufacturer, the
detection limit of the assay (minimum measurable S100B value,
B0 value +3 SD) was 0.02 µg/L, and the
within- and interassay precisions (CV) were 
5.5% and 10%,
respectively, for concentrations at 0.28–4.17 µg/L.
S100B concentrations were expressed as mean values ± SD. Spearman rank-order correlation and comparison were determined between groups by Kruskal–Wallis one-way ANOVA and the Mann–Whitney U-test when data did not have a gaussian distribution. The sensitivity and specificity of urinary S100B measurement as a diagnostic test was assessed by a ROC curve test (15). Statistical significance was set at P <0.05.
At birth, there was no difference in the mode of delivery, weeks of gestation, weight, Apgar score at 1 and 5 min, the incidence of acute respiratory distress syndrome, clinical and laboratory results, cerebral ultrasound scans, and neurologic examination between preterm infants with and without IVH. In this regard, urea, creatinine clearance, osmolarity, and urinary specific weight in the two groups were superimposable. At the 24-h time point, in all but two infants (IVH grade I hemorrhage), cerebral ultrasound examination was negative for IVH.
S100B concentrations in control preterm infants at the first urination
were within the reference interval, as described previously
(10). In the newborns who developed IVH, urine S100B was
significantly higher than in controls at all monitoring time points
(P <0.001 for all), increasing progressively from first
urination to 72 h (Fig. 1A
). A positive correlation was also found between S100B concentrations
measured at the first urination and IVH grade, radiologically defined
at 72 h, indicating the extent of the brain lesion
(r2 = 0.76; P <0.001; Fig. 1B
). When infants were subgrouped according to ultrasound scanning
patterns, urine S100B at birth was significantly higher in the
following infants than in controls (0.29 ± 0.18 µg/L): infants
who developed IVH with parenchymal lesion (3.51 ± 0.33 µg/L) at
72 h after birth; infants with IVH associated with
ventriculomegally (2.66 ± 0.18 µg/L); and infants with IVH
alone (1.81 ± 0.56 µg/L; P <0.001 for all). In
addition, a significant difference in S100B concentrations was observed
among the three IVH groups (P <0.001 for all). The
sensitivity and specificity of the S100B protein as a diagnostic test
were 100% (95% confidence interval, 80.5–100%) at a urine
S100B cutoff of 0.70 µg/L.
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The five infants who died after the first 72 h showed the highest S100B concentrations.
The potential usefulness of this finding derives from the consideration that it could offer a measurable indicator of brain lesion that correlates significantly with the degree of brain hemorrhage at a point when radiologic and clinical assessments are of no avail. In addition, the significantly higher concentrations of urine S100B in IVH infants without radiologically apparent ventriculomegally or parenchymal lesions could reasonably be a biochemical marker indicating a subclinical lesion. The finding also constitutes the first observation of S100B in urine as a marker of brain damage. The increased concentrations of S100B in urine are not surprising because urinary S100B can be reasonably supposed to originate directly from the blood, where it has already been shown to be increased in IVH infants (9). It should be noted that S100A1 (formerly called S100A0), which is muscular in origin but very similar to S100B in structure and molecular weight, has already been shown to exhibit the same patterns of concentration in the blood and urine of patients subjected to open-heart surgery (16). In addition, because renal function also appears to be normal in preterm infants, the different S100B urine concentrations observed in preterm newborns cannot reasonably be ascribed to different degrees of urine concentration. Because S100B is known to be essentially absent from kidney tissue, which, on the contrary, is rich in S100A1 (17)(18), the original source of S100B in the urine could be the fetal nervous system, which is known to contain the protein at the stages of development under examination (19)(20). S100B may also be released from other sites of concentration, such as adipose tissue (21), although its adipocyte location at this age has not been conclusively studied. The phenomenon could also be related to a biologic role exerted by S100B. On the basis of findings in experimental models and in human studies, the protein has been proposed to act as a cytokine with a neurotropic effect at physiologic concentrations (3)(10)(22)(23) but to exert a neurotoxic role at high concentrations (3)(24). The possibility that at least part of the S100B measured in the urine of these patients derives from this process, which may be part of a cascade of pathologic events accompanying parenchymal damage, should be taken into consideration.
Our results are in agreement with recent data (25) indicating a relationship between increased S100B in the CSF of preterm infants and the degree of brain injury in the presence of parenchymal damage, although these authors found no significant differences between concentrations of the protein in the CSF of IVH infants without radiologically apparent parenchymal damage and controls. This discrepancy could be explained by the differences in monitoring procedures (longitudinal urine sampling from birth to 72 h, corresponding to ultrasound diagnosis of IVH in our study vs one CSF sample performed in infants when posthemorrhagic ventricular dilatation was clearly detectable by ultrasound).
In conclusion, although increased S100B in the blood has already been shown to constitute an early indicator of IVH in preterm infants (9), for the first time, the use of S100B as a pathologic marker in urine, which is much more easily tested than blood or CSF, offers a new perspective for the study of S100B in biologic fluids. This is especially promising in view of the possibility of improving the care of newborns because anemia in premature infants caused by blood sampling is a common pathology (26).
Acknowledgments
This work was partially supported by grants from Università Cattolica del S. Cuore and from Ministero dell’Università e Ricerca Scientifica e Tecnologica (to F. M.) and from the "Let’s Improve Prenatal Life" Foundation (to D. G.). We also thank Sangtec, Medical (Bromma, Sweden) and Byk Goulden Italia for supplying reagent sets. We would also like to thank the Nursery Team of the Department of Pediatrics of the Giannina Gaslini Children’s University Hospital, Genoa, for their enthusiastic cooperation during this research project.
References
and S100-ß proteins in normal nonnervous human tissues. Lab Invest 1987;57:489-498.[ISI][Medline]
[Order article via Infotrieve]
-like immunoreactivity in tubules of rat kidney. A clue to the function of a "brain-specific" protein. J Histochem Cytochem 1985;33:367-374.[Abstract]
The following articles in journals at HighWire Press have cited this article:
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  A Pickering, J Carter, I Hanning, and W Townend Emergency department measurement of urinary S100B in children following head injury: can extracranial injury confound findings? Emerg. Med. J., February 1, 2008; 25(2): 88 - 89. [Abstract] [Full Text] [PDF]
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 D. Gazzolo, E. Marinoni, R. Di Iorio, M. Lituania, M. Marras, M. Bruschettini, P. Bruschettini, R. Frulio, F. Michetti, F. Petraglia, et al. High Maternal Blood S100B Concentrations in Pregnancies Complicated by Intrauterine Growth Restriction and Intraventricular Hemorrhage Clin. Chem., May 1, 2006; 52(5): 819 - 826. [Abstract] [Full Text] [PDF]
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 D. Gazzolo, E. Marinoni, R. Di Iorio, M. Bruschettini, M. Kornacka, M. Lituania, U. Majewska, G. Serra, and F. Michetti Measurement of Urinary S100B Protein Concentrations for the Early Identification of Brain Damage in Asphyxiated Full-term Infants Arch Pediatr Adolesc Med, December 1, 2003; 157(12): 1163 - 1168. [Abstract] [Full Text] [PDF]
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F. Michetti and D. Gazzolo S100B Protein in Biological Fluids: A Tool for Perinatal Medicine Clin. Chem., December 1, 2002; 48(12): 2097 - 2104. [Abstract] [Full Text] [PDF]
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D. Gazzolo, M. Bruschettini, R. Di Iorio, E. Marinoni, M. Lituania, M. Marras, R. Sarli, P. L. Bruschettini, and F. Michetti Maternal Nitric Oxide Supplementation Decreases Cord Blood S100B in Intrauterine Growth-retarded Fetuses Clin. Chem., April 1, 2002; 48(4): 647 - 650. [Full Text] [PDF]
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) and control (•) groups expressed as mean ± SD at the
following time points: first urination (0), 24 h
(1), 48 h (2), and 72 h
(3). S100B concentrations were significantly higher at
all monitoring time points (P <0.001) in the IVH group.
(B), correlation between urinary S100B measured at birth
(
indicates deceased infants.