Study of perinatal asphyxia and its outcome concerning nucleated RBC count in venous blood of term neonates

Introduction: Perinatal asphyxia is a serious problem globally and is one of the common causes ofneonatal mortality. Worldwide each year four million infants suffer from birth asphyxia. Of these onemillion die and an equal number develop serious sequelae. Perinatal asphyxia ranks as the secondmost important cause of neonatal death after infections accounting for about 23% of mortalityworldwide. Aim & objective: To establish the level of nucleated red blood cells as an indicator ofpoor immediate outcome in perinatal asphyxia. Method: It was a case-control study done in thedepartment of pediatrics, S C B Medical College & S V P PG I P, Cuttack. Observation: The NRBCcount was significantly higher in neonates with adverse outcomes than in those with favourableoutcomes (p-value <0.001). NRBC count cut-off of >27/100 WBC had a sensitivity of 75% andspecificity of 95.2% in predicting adverse outcome defined as death, hemiplegia, hypertonia orsignificant hypotonia, unreliable sucking and seizures resistant to Phenobarbital. Conclusion:Nucleated red blood cell count can be used as a surrogate marker for birth asphyxia. It has asignificant negative correlation with Apgar score at one minute and Apgar score at 5minutes &significant positive correlation with severity of hypoxic ischemic-encephalopathy, time is taken forrecovery of neurological impairment following birth asphyxia and duration of NICU stay.


Introduction
Birth asphyxia is a serious clinical problem worldwide and contributes greatly to neonatal mortality and morbidity [1]. The incidence of birth asphyxia is about 1-1.5% of live births in most centres and inversely related to gestational age, and birth weight, lower considerably in later gestation [2]. Studies have been done to correlate many nucleated red blood cells (NRBC) in cord blood with perinatal asphyxia and they have shown a good correlation [10,11,12,13].
An increase in NRBC has been proposed due to increased production of erythropoietin secondary to perinatal asphyxia [14]. Most studies in the literature have used cord blood to report NRBC count. In developing countries like India, it is often difficult to collect cord blood as a large number of deliveries takes place in peripheral health centres or at home. NRBC count in venous blood can be a costeffective and logistically plausible alternative to cord blood NRBC. Hence the present study is intended to evaluate the significance of the presence of NRBC per 100 WBC in venous blood in the prediction of perinatal asphyxia and its outcome; and to compare this with established markers such as Apgar score.

Aim & objective
To compare the levels of nucleated red blood cells in the venous blood of asphyxiated newborns with non asphyxiated newborns & establish the level of nucleated red blood cells as an indicator of poor immediate outcome in perinatal asphyxia. according to NRBC count. The first group had an NRBC count of 0-9, the second had an NRBC count of 10-19 and the third group had an NRBC count of 20 and more. To compute the difference between the asphyxiated and the non-asphyxiated group, Student t-test, Chi square-test and one-way ANOVA was applied as appropriate. The receiver operating curve (ROC) was used to derive the cut-off value for NRBC count for predicting perinatal asphyxia as well as to predict adverse outcome in perinatal asphyxia.

Methodology
A p-value of <0.05 was considered statistically significant. The results are presented as means (± S.D.) unless otherwise indicated. All data were statistically analyzed using SPSS statistics software for Windows (Version 20).    The majority of the cases had HIE stage 2(52%).

Observationv
HIE stage 3 was seen in 24% of the cases. The remaining 12 cases (24%) had HIE stage 1 Both study groups were similar in terms of the total leukocyte count. But platelet count was significantly lower in cases compared to controls (p-value 0.010).
In the present study, statistically significant higher NRBC counts were seen in the perinatal asphyxia group in comparison to the control group(p-value<0.001).  NRBC count was higher in cases compared to statistically significant controls.
(P-value<0.001) Nucleated RBC count increased with decreasing Apgar score at both one minute and 5 minutes. Among the cases, a statistically significant negative correlation exists between NRBC count and Apgar score at1-minute as well as at 5-minutes. No such correlation was found among the controls.
Among the asphyxiated babies, a statistically significant positive correlation exists between NRBC count and time taken to start direct breast feeding, Recovery of neonatal reflexes, recovery of good cry, suck and activity, duration of stay in NICU and total hospital stay. The mean time is taken to start direct breastfeeding, recovery of neonatal reflexes, recovery of a good cry, suck and activity, duration of stay in NICU and total hospital stay was longest in group 3 followed by group 2 and was least in group 1. This was statistically significant for all immediate outcomes studied except for recovery of neonatal reflexes and recovery of a good cry, suck and activity. There was a total of 8 deaths in the study. All deaths occurred in the cases with NRBC count 20 and more.47%of total cases with NRBC count of 20 or more died. This group also had a significantly longer hospital stay compared to other groups which was statistically significant (p-value0.001)

Discussion
In the present study there were 50 babies in both study groups. There was no significant difference in the sex distribution of babies between the two study groups. The two study groups were similar in terms of age and parity of the mother. The mean birth weight of babies in the case group was nearly 130 gms more than the mean birth weight of the babies in the control group, but this difference was not statistically significant (p=0.071). Gestational age was higher in cases compared to controls, which was statistically significant.
62.5% of cases were associated with meconiumstained amniotic fluid. Other risk factors associated with birth asphyxia were a cord around the neck, the prolonged second stage of labour, secondary arrest in the descent of the fetus and short stature.
There was no statistically significant difference in the mode of delivery between the two study groups although more number of babies with birth asphyxia were born of assisted (forceps) vaginal delivery.
In the present study, the NRBC count for normal neonates was found to be 3.8± 3.09 NRBC/100WBC, which was consistent with previous reports [15,16,17]. NRBC count/100leukocytes increased during the first few hours of life in perinatal asphyxia as compared with healthy control subjects. The mean NRBC count in asphyxiated babies was 20.3 (±4.47) NRBC/100WBC, ranging from 7 to 93. Hence, the NRBC count was significantly higher in the asphyxiated group compared to normal babies (p-value < 0.001).
The mechanism causing the rapid release of NRBC following perinatal asphyxia is not known, although increased erythropoietin results from hypoxia and probably has a major role in the process [17,18,]. NRBCs are immature erythrocytes whose production is thought to be driven primarily by the interplay of hypoxia and erythropoietin (EPO) synthesis [14].
Previous studies suggest that EPO increases erythroid production and releases immature forms of erythrocytes into the peripheral circulation in response to hypoxia. It is possible that increased NRBC production in the immediate neonatal state primarily reflects hypoxic injury [19]. Several studies have reported an increased NRBC in neonatal blood following perinatal asphyxia [16,18,20,21,22,23].
Boskabadi et al [16] reported a cut-off value for NRBC as>70/mm3 with a sensitivity of 83.4% and specificity of 73.5% in predicting perinatal asphyxia.
The results of the present study give additional support to previous reports and also defines the cutoff value for NRBC as >9/100WBC with a sensitivity of 96% and specificity of 94% in predicting birth asphyxia.
Neonates diagnosed with HIE were found to have higher NRBC counts, when compared with control infants. NRBC count was significantly related to the Nucleated red blood cells are commonly seen in the cord blood of healthy newborns at birth. In term non-asphyxiated newborns, the number of nucleated red blood cells is variable but is only rarely higher than10/100WBC. The mechanism causing the rapid release of NRBC following perinatal asphyxia is not known, although increased erythropoietin results from hypoxia and probably has a major role in the process [17,18].
Several studies have reported an increased NRBC in neonatal blood following perinatal asphyxia [16,18,20,21,22,23]. Boskabadi et al [16] reported a cut-off value for NRBC as>70/mm3 with a sensitivity of 83.4% and specificity of 73.5% in predicting perinatal asphyxia. The results of the present study give additional support to previous reports and also defines the cut-off value for NRBC as >9/100WBC with a sensitivity of 96% and specificity of 94% in predicting birth asphyxia. In the present study there were 50 babies in both study groups. There was no significant difference in the sex distribution of babies between the two study groups. The two study groups were similar in terms of age and parity of the mother. The mean birth weight of babies in the case group was nearly 130 gms more than the mean birth weight of the babies in the control group, but this difference was not statistically significant (p=0.071).
Gestational age was higher in cases compared to controls, which was statistically significant. 62.5% of cases were associated with meconium-stained amniotic fluid. Other risk factors associated with birth asphyxia were a cord around the neck, the prolonged second stage of labour, secondary arrest in the descent of fetus and short stature.
There was no statistically significant difference in the mode of delivery between the two study groups although more number of babies with birth asphyxia was born of assisted (forceps) vaginal delivery. Babies born of emergency LSCS were excluded from both study groups.
In the present study, the mean (± SD) NRBC count in normal term newborns born of normal vaginal delivery was 3.8 ± 3.09 NRBC/100WBC (range 0-16

Limitation of the study
The present study includes only the baby born in term & by normal vaginal route. So the result could not be generalized to the newborn population. The study was limited up to discharge; so long term outcome could not be determined.

Conclusion
Nucleated red blood cell count can be used as a surrogate marker for birth asphyxia. It has a significant negative correlation with Apgar score at one minute and Apgar score at 5minutes & significant positive correlation with severity of hypoxic ischemic-encephalopathy, time is taken for recovery of neurological impairment following birth asphyxia and duration of NICU stay. The NRBC cutoff value of >9/100 WBC has a sensitivity of 96% and specificity of 94% in predicting birth asphyxia, defined as Apgar at 1 minute ≤ 6/10. NRBC count cut-off of >27 per 100 WBCs has a sensitivity of 75% and specificity of 95.2% in predicting adverse short term outcome including early neonatal death in perinatal asphyxia.
What does this study add to the existing knowledge?
Nucleated RBC count in peripheral venous blood is a low cost, simple and easily available test which can be done in any health care facility with minimal infrastructure.