|Year : 2017 | Volume
| Issue : 2 | Page : 588-594
Umbilicalcord S100B protein in neonatal hypoxic–ischemic encephalopathy
Ghada M El-Meshad1, Dalia M El-Lahoney1, Naglaa F Barseem1, Mohammed A Helwa2, Amr A.H. Elsisy3
1 Department of Pediatrics, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Department of Pediatrics, El Shohadaa Central Hospital, Menoufia, Egypt
|Date of Submission||30-Nov-2016|
|Date of Acceptance||01-Mar-2017|
|Date of Web Publication||25-Sep-2017|
Amr A.H. Elsisy
Faculty of Medicine, Menoufia University, Shebin Elkoom, Menoufia Governorate, 32511
Source of Support: None, Conflict of Interest: None
The aim of this study was to highlight the importance of cord blood S100B protein in the diagnosis of neonatal hypoxic–ischemic encephalopathy and determination of its severity.
S100B is a calcium-binding protein and is a major component of the cytosol in various cell types. The S100B exerts significant influence on cellular metabolism, Ca2+ homeostasis, cytoskeletal modification, cell proliferation, and cell differentiation. The presence of S100B in the cerebrospinal fluid, serum, and amniotic fluid above threshold levels is used for diagnostic/prognostic purposes.
Patients and methods
This study included 30 asphyxiated newborns and 23 weight and gestational age-matched healthy neonates as controls. Immediately after birth, blood samples were collected from all neonates and values of S100B protein were determined using enzyme-linked immunosorbent assay technique.
The mean serum level of S100B protein was significantly higher in the asphyxiated group than in the control group with a significant correlation between increased S100B protein level and severity of hypoxic–ischemic insult among patients. We found that at the cutoff level for serum S100B protein of 0.44 μg/l, the sensitivity was 97%, specificity was 91%, and accuracy of predicting neonatal asphyxia was 94%, with a positive predictive value of 94% and a negative predictive value of 95%.
It was concluded that S100B protein in the umbilical cord blood is a useful marker for early detection of neonatal hypoxic–ischemic encephalopathy in the full-term neonate and also in determining the grade of hypoxia.
Keywords: encephalopathy, ischemia, neonatal hypoxia, S100B protein
|How to cite this article:|
El-Meshad GM, El-Lahoney DM, Barseem NF, Helwa MA, Elsisy AA. Umbilicalcord S100B protein in neonatal hypoxic–ischemic encephalopathy. Menoufia Med J 2017;30:588-94
|How to cite this URL:|
El-Meshad GM, El-Lahoney DM, Barseem NF, Helwa MA, Elsisy AA. Umbilicalcord S100B protein in neonatal hypoxic–ischemic encephalopathy. Menoufia Med J [serial online] 2017 [cited 2020 Jan 22];30:588-94. Available from: http://www.mmj.eg.net/text.asp?2017/30/2/588/215474
| Introduction|| |
Neonatal encephalopathy is the clinical phenotype characterized by a syndrome of disturbed neurologic function in the earliest days of life in term infants, manifested by difficulty with initiating and maintaining respiration, depression of tone and reflexes, subnormal level of consciousness, and often by seizures . The incidence in developed countries is estimated to be 1.5 per 1000 live births , and estimates in developing countries range from 2.3 to 26.5 per 1000 live births .
Hypoxic–ischemic encephalopathy (HIE) may be due to any condition leading to decreased oxygen supply (hypoxia) and decreased blood supply (ischemia). With hypoxia and ischemia, the brain converts to anaerobic metabolism, resulting in the depletion of adenosine triphosphate and accumulation of lactic acid and free radicals . In term neonates, 90% of asphyxia insults occur during the antepartum or intrapartum period as a result of placental insufficiency, and the remaining 10% occurs postpartum, usually secondary to pulmonary, cardiovascular, or neurologic disorders .
The pathway of cerebral injury in the term infant with HIE is not always clear. Many factors, including the etiology, extent of hypoxia or ischemia, maturational stage of the brain, regional cerebral blood flow, and general health of the infant before the injury, can all impact the pattern and extent of brain injury as well as the outcome following injury .
The development of brain injury after hypoxic–ischemic insult is an evolving process imitated during the acute insult and extending into a reperfusion phase. The principle pathogenic mechanism underlying neurological damage in HIE resulting from hypoxemia/ischemia or both is deprivation of glucose and oxygen supply, which causes a primary energy failure and initiates a cascade of biochemical events leading to cell dysfunction and ultimately to cell death .
The diagnosis of perinatal asphyxia is primarily based on assessment of the general health of the infant (Apgar score), confirmation of the presence of a metabolic acidosis (cord blood pH and base deficit), and the need for resuscitation. If an episode of asphyxia results in central nervous system dysfunction, this neuronal injury is called neonatal encephalopathy .
In 1976, Sarnat and Sarnat designed a score, the Sarnat score, which was a combination of neurological symptoms, as described above, and the accompanying electroencephalographic features. This score was in fact a grading of the severity of the encephalopathy caused by the perinatal hypoxia–ischemia. It contains three stages, HIE stage 1, HIE stage 2, and HIE stage 3, the last stage being the most severe stage of encephalopathy. The most severely affected infants typically progress from stage 1 to stage 3 .
Asphyxia can lead to multiorgan dysfunction and a redistribution of cardiac output to maintain cerebral, cardiac, and adrenal perfusion while potentially compromising renal, gastrointestinal, and skin perfusion .
The aim of this study was to highlight the importance of cord blood S100B protein in the diagnosis of neonatal HIE and determination of its severity.
| Patients and Methods|| |
This cross-sectional study was conducted on 30 asphyxiated full-term neonates delivered in Shebin El-Kom Teaching Hospital during a period of 6 months from January 2016 to June 2016. These were compared with 23 age-matched apparently healthy term neonates as a control group with no obstetrical problems.
Inclusion criteria were as follows: profound metabolic or mixed acidosis in an umbilical artery blood sample; persistence of an Apgar score of 0–3 for longer than 5 min; neonatal neurologic complications (e.g., seizures and coma); and multiple organ involvement (e.g., the kidney, lung, liver, heart, and intestines) .
Exclusion criteria were as follows: prematurity, traumatic brain injuries, maternal drug addiction, major congenital malformation, Down's syndrome, intrauterine infection, and general anesthesia during birth process.
All cases and controls were subjected to the following: full maternal history with special emphasis on medical and obstetric data at delivery, including the mode of delivery; Apgar score at 1 and 5 min and resuscitation data; assessment of gestational age using the Ballard scoring system; anthropometric measurements (head circumference, weight, and length), evaluation of vital signs and socioeconomic status; full systemic examination; and neurological examination with the assessment of severity of HIE using Sarnat's staging.
Serum sampling and determination of S100B protein level
Cord blood samples were collected as early as possible immediately after birth and analyzed for arterial blood gases. Blood film was prepared to measure the level of nucleated red blood cells (RBCs). A volume of 1 ml of cord blood sample was collected from all neonates included in the study, and it was centrifuged at 2500 rpm. for 5 min and frozen at –80°C for a maximum of 6 months. S100B protein level was measured in these samples.
The kit was based on standard sandwich enzyme-linked immunosorbent assay technology. The purified anti-S100B antibody was precoated onto 96-well plates, and the horseradish peroxidase-conjugated anti-S100B antibody was used as detection antibodies. The standards, test samples, and HRP-conjugated detection antibody were added to the wells, and mixed and incubated; thereafter, unbound conjugates were washed away with wash buffer. tetramethylbenzidine (TMB) substrates (A and B) were used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that changed to yellow color after adding acidic stop solution. The density of the yellow product is proportional to the S100B amount of sample captured in plate. The optical density absorbance was read at 450 nm in a microplate reader, and then the concentration of S100B was calculated (Chongqing Biospes Co. Ltd Chongqing, China).
This study has been approved by the ethical committee of Faculty of Medicine, Menoufia University. Moreover, informed consent from parents of each studied neonate was obtained.
| Results|| |
In the present study, there was no significant difference between the asphyxiated and the nonasphyxiated groups with regard to gestational age, birth weight, sex consanguinity, socioeconomic status, and mode of delivery [Table 1]. According to our results, the maternal history included premature rupture of membranes, antepartum hemorrhage, pre-eclampsia, and history of previous pregnancies, delivery, or abortion, but they were not significant risk factors for the increased incidence of HIE [Table 2]. In our study, there was a highly significant difference between the two studied groups with regard to clinical manifestations: cyanosis, 21 cases in the asphyxiated group; abnormal resting posture (distal flexion and frog leg position), 16 cases in the asphyxiated group; abnormal consciousness (stupor, lethargy, and comatose), 19 cases in the asphyxiated group; abnormal muscle tone (hypotonia and flaccidity), 18 cases in the asphyxiated group; poor or absent suckling reflex, 24 cases in the asphyxiated group; absent Moro reflex, 19 cases in the asphyxiated group; and convulsions, 18 cases in the asphyxiated group (P < 0.001). Other clinical manifestations showed a significant difference between the two groups: abnormal respiratory pattern (rapid deep, rapid shallow, and apnea), 16 cases in the asphyxiated group, and grunting with respiration, nine cases in the asphyxiated group (P = 0.001). Other clinical manifestations showed no significant difference between the two groups, such as pallor or birth injuries (P > 0.05) [Table 3]. In the present study, Apgar scores at 1 and 5 min were significantly lower in cases (with mean values of 2.07 ± 0.83 and 3.97 ± 0.89, respectively) when compared with controls. As regards arterial blood gases, our results showed that the mean pH (7.11 ± 0.11), PaO2 (55.71 ± 9.41 mmHg), PaCO2 (37.29 ± 5.32 mmHg), HCO3 (13.51 ± 2.38 mmol/l), and base excess (BE) (14.17 ± 2.13 mmol/l) were significantly lower in hypoxic newborns when compared with the control group. The present study showed that the mean serum level of S100B protein was significantly higher in the HIE group (0.72 ± 0.17 μg/l) compared with the control group (0.24 ± 0.14 μg/l), and the percentage of cases that had nucleated RBCs in the blood was 70.6% in comparison with controls (29.4%) [Table 4]. In addition, there was a strong correlation between increased concentrations of S100B protein in serum and the severity of HIE in the patient group [Table 5]. This study demonstrated a highly significant negative correlation between serum S100B protein level and Apgar score at 1 and 5 min. Moreover, there was a highly significant negative correlation between serum S100B protein level and arterial pH and PaO2, whereas a highly significant positive correlation was found between serum S100B protein and base excess [Table 6]. According to our results, the best cutoff value of serum S100B protein in predicting HIE was 0.44 μg/l (P < 0.001), with a sensitivity of 97%, specificity of 91%, positive predictive value (PPV) of 94%, negative predictive value (NPV) of 95%, and diagnostic accuracy of 94% [Figure 1]. Moreover, our results showed that the best cutoff value for nucleated red blood cells (NRBCs) was 5 NRBCs/100 white blood cells (WBC) to detect hypoxia with a sensitivity of 80%, specificity of 78%, PPV of 83%, NPV of 75%, and diagnostic accuracy of 79% [Figure 2].
|Figure 1: Receiver operating characteristic (ROC) curve to determine the sensitivity and specificity of serum S100B protein with a cutoff point of 0.44 μg/l to diagnose hypoxic–ischemic encephalopathy. Area under curve: 0.967.|
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|Figure 2: Receiver operating characteristic (ROC) curve to determine the sensitivity and specificity of nucleated red blood cells (normoblasts) with cutoff point of 5 nucleated red blood cells/100 white blood cells to diagnose hypoxic–ischemic encephalopathy. Area under curve: 0.806.|
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|Table 1: Sociodemographic data, anthropometric measures, and mode of delivery of the studied groups|
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|Table 2: Comparison between the studied groups as regards their maternal history|
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|Table 3: Comparison between the studied groups as regards their clinical picture|
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|Table 4: Comparison between the two groups as regards Apgar score and laboratory investigations|
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|Table 5: Comparison between the Sarnat stages as regards serum level of S100B protein (n=30)|
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|Table 6: Correlation between S100B protein level and arterial blood gas and Apgar score at 1 and 5 min among cases|
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| Discussion|| |
HIE remains a serious condition, causing significant mortality and long-term morbidity. Despite major advances in monitoring technology and knowledge of fetal and neonatal pathologies, perinatal asphyxia remains a major concern .
The mechanism of damage from hypoxic–ischemic insult involves a series of events such as a decrease in intracellular energy, a decrease in membrane Na + and K +-ATPase activity, an increase in intracellular Ca 2+, and membrane lipid peroxidation .
There are some studies on S100B protein level as a marker for HIE and evaluation of S100B protein level in different HIE clinical stages caused by perinatal asphyxia.
The aim of the present study was to highlight the importance of cord blood S100B protein in the diagnosis of neonatal HIE in full-term neonates and thus could be marker in these cases.
In our results there was no statistically significant difference between the HIE and control groups as regards sex, mode of delivery, weight, and gestational age.
There was no sex difference as regards hypoxic insult. This is in accordance with that reported by other authors (Tekgul et al.  and Kirimi et al. . However, Futrakual et al.  found a significant relation between HIE and male sex in their study as risk factors for HIE.
There was no statistically significant difference between the HIE and the control group as regards the mode of delivery, and this is in accordance with other authors ,,,. However, Kaye  and Zhang et al.  stated that cesarean section was highly associated with HIE, and Butt et al.  found that HIE developed in 76.5% of vaginal delivered cases, whereas cesarean section occurred in 23.5% of cases. The variation in different studies concerning mode of delivery may be attributed to the fact that neonatal encephalopathy may originate early in the antepartum period in some cases of HIE.
There was no statistically significant difference between the HIE and the control group as regards gestational age. This is in accordance with the findings of Vasiljevic et al. . Futrakual et al.  stated that gestational age, particularly post-term gestation, is significantly associated with HIE; this might be related to the uteroplacental insufficiency.
The current study showed no statistically significant difference between the HIE and control groups as regards birth weight. This is in accordance with the findings of Ghotbi and Najibi , but Aired  reported that infants with intrauterine growth retardation play a significant role in the occurrence of severe asphyxia due to placental insufficiency. This conflict may be attributed to deprivation of our sample from cases with intrauterine growth retardation.
In the present study, the median of Apgar score at 1 and 5 min was 2 and 4, respectively, and it was significantly lower than that in the control group, which had a normal Apgar score (6–10 at 1 and 5 min). Cases showed a statistically significantly low Apgar score compared with controls, and this is in agreement with the findings of Karlsson et al. .
In the present study, we found that nucleated RBCs can be a reliable marker to detect hypoxia, but there are many markers superior to it as its sensitivity was 80%, specificity was 78%, PPV was 83%, NPV was 75%, and diagnostic accuracy was 79% with a cutoff point of 5 NRBCs/100 WBC. This is in accordance with the findings of Boskabadi et al. , who showed a sensitivity of 83.4% and a specificity of 73.5% for nucleated RBCs. Moreover, Lailah et al.  found that nucleated RBC count was reliable in detecting HIE as area under the curve was 84% and the best cutoff for nucleated RBC in diagnosing hypoxia was greater than 4.5/100 WBCs with a sensitivity of 77%, specificity of 83%, PPV of 82%, and NPV 78% with a diagnostic accuracy of 80%.
In the present study, S100B protein levels were significantly higher in hypoxic than in healthy newborns. This is in accordance with the findings of Beharier et al.  and Douglas-Escobar and Weiss .
The mean S100B protein in the asphyxiated group was 0.72 μg/l, whereas the mean S100B protein in the control group was 0.24 μg/l. This is in agreement with the findings of Sofijanova et al. , who found that the mean S100B protein in asphyxiated newborn was 0.642 μg/l, and in the control group the mean S100B was 0.119 μg/l. In addition, S100B protein cutoff point was 0.12 μg/l for predicting HIE. This is in agreement with the present study, in which S100B protein cutoff point was 0.44 μg/l. Qian et al.  found that umbilical cord arterial blood concentration of S100B more than 2.20 μg/l has a sensitivity of 87% and a specificity of 88% in predicting moderate-to-severe HIE. The lack of agreement between the study by Qian et al.  and the present study may be attributed to the following: first, the two umbilical arteries transport metabolites from the fetus back to the placenta, and hence the biochemical markers in them can reflect the condition of the fetus more accurately; second, it can be attributed to environmental factors and individual variations between western and middle east people.
In the present study serum S100B protein level showed a sensitivity of 97% and specificity of 91%, PPV of 94%, and NPV of 95% with a diagnostic accuracy of 94%, which is superior to other markers such as lactate, which showed a sensitivity of 94% and specificity of 87% .
Moreover, S100B protein is superior to other markers such as NRBCs, which showed a sensitivity of 83.4% and specificity of 73.5% .
High S100B protein sensitivity and specificity values were observed in the present study for identifying HIE in full-term neonates with low Apgar score or the need for cardiopulmonary resuscitation at birth. These data are important for supporting early diagnosis and being able to opportunely initiate therapeutic measures for preventing mortality and long-term neurological consequences.
The present study compared S100B protein results with the different clinical stages of the Sarnat clinical classification for HIE diagnosis and showed that there was a positive correlation between HIE stage and S100B protein level. This is in accordance with the findings of Qian et al. . However, Nagdyman et al.  found that asphyxiated term infants had elevated S100B protein levels in their umbilical vein blood, but no difference was observed between mild and moderate or severe HIE.
Moreover, we found that there were significant correlations between umbilical cord blood S100B protein and PH, BE, and Apgar score at 1 and 5 min. This is in agreement with the findings of Qian et al. , who stated that there was a close correlation between the S100B protein in arterial cord blood and pH (r=−0.74, P < 0.001), and the correlation was even stronger between S100B protein and BE (r=−0.79, P < 0.001). As expected, S100B protein also correlated significantly to the Apgar score at 1 min (r=−0.65, P < 0.001) and the Apgar score at 5 min (r=−0.72, P < 0.001).
| Conclusion|| |
The measurement of S100B protein in the umbilical cord blood is rapid, noninvasive, inexpensive, and simple to perform, and could possibly become a useful marker for early detection of HIE in the full-term neonate and also in determining the severity of the disease and subsequently the short-term outcome.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]