Nucleated Red Blood Cell in Cord Blood as a Marker of Perinatal Asphyxia

Address for correspondence: Dr. Piush Kanodia DM Neonatology Resident Department of Paediatrics BPKIHS, Dharan, Nepal Email: piushkanodia@yahoo.com Mobile no: +9779858024990 1Dr. Piush Kanodia, Neonatology resident, Division of Neonatology. 2Prof Nisha Keshary Bhatta, Professor, Division of Neonatology. 3Prof Rupa Rajbhandari Singh, Chair, Division of Neonatology. 4Prof Gauri Shankar Shah, Professor and Head. 5Dr. Shankar Prasad Yadav, Assistant Professor. 6Dr. Sunil Kumar Yadav, Neonatology resident, Division of Neonatology. All from the Department of Paediatrics and Adolescent Medicinem, B.P Koirala Institute of Health Sciences, Dharan, Nepal. Abstract


Introduction
P erinatal asphyxia is a common problem with the incidence varying from 0.5 -2% of live births 1,2,3,4 .The highest annual neonatal deaths are in South Asia, where an es mated 51 deaths occur for every 1,000 live births 5 amongst which 21.0% of newborn mortality occurs due to birth asphyxia 5 .Failure to ini ate and sustain breathing immediately a er delivery has been associated with hypoxic ischemic injury to the central nervous system (CNS) and the clinical manifesta ons of this injury have been termed as Hypoxic Ischemic Encephalopathy (HIE).Nucleated Red Blood Cell (NRBC) counts in umbilical blood of neonates have been reported as a possible marker of perinatal asphyxia 6 .The number of NRBC per 100 WBC is variable but is rarely greater than 10 in normal neonates 6,7,8,9,10,11 .The hypoxic event induces a compensatory response in the form of exaggerated erythropoesis, resul ng in the release of immature red blood cells into the fetal circula on.
The aim of the study was to fi nd out the associa on of nucleated RBC with perinatal asphyxia, HIE and Apgar score

Material and Methods
This prospec ve case-control study was performed on 164 term newborns, out of which 82 were cases and rest 82 were control.This study was conducted at Neonatology division of BPKIHS.Study period was for one year from May 2014 to may 2015.The cases were selected as per WHO defi ni on-"failure to ini ate and sustain breathing at birth," Apgar score of 6 or less at 5 minute, Neonatal arterial blood gas pH < 7.2 or a base defi cit of at least 12 mmol/lit with in the fi rst hour of life, fetal distress, thick, meconium stained amnio c fl uid and resuscita on for more than 1 minute with posi ve pressure ven la on.Babies with major congenital malforma ons, preterm and born through general anesthesia and Magnesium sulphate for Eclampsia were excluded from the study.Wri en informed consent was obtained from the parents before the commencement of study.Detailed antenatal, natal and postnatal history was obtained to elicit evidence of perinatal asphyxia and clinical examina on of the newborn was performed to assess the gesta onal age (as per New Ballard score) and following which detailed systemic examina ons was conducted.The clinical severity and progress of asphyxial insult was evaluated on a Classifi ca on of HIE (Sarnats and Sarnats).
Relevant inves ga ons were carried out inves ga ons as and when required: like Arterial Blood Gas, Nucleated RBC, Haemoglobin, Total Leucocyte Count, Diff eren al Leucocyte Count, Platelet and Re culocyte count, peripheral smear for General Blood Picture, blood urea, serum electrolyte, serum crea nine, sepsis, screen (absolute neutrophil count, band cell ra o, micro ESR, CRP) and blood culture and sensi vity.Cytological and biochemical evalua on of cerebrospinal fl uid including culture and sensi vity was done.For Nucleated RBC count (case and control group), immediately a er delivery, 1 mL of umbilical cord venous blood was collected in a tube containing 1.5 mg ethylenediaminetetra ace c acid (EDTA) and cord venous blood was dispatched to central pathology laboratory.Thin blood fi lm was prepared from umbilical blood.The fi lm was stained by Leishman's stain and nRBC/ 100 WBC was counted under pathologist's supervision.
The study was started a er the approval of the Ins tu onal Ethical Review Board.A wri en en informed consent was obtained from each parent of study subjects.The par cipants had op on to withdraw from the study any me during their hospital stay.Data were analyzed using the SPSS version 20.0.Chi-square and Fisher Exact tests were applied to compare the data of propor ons and Student's t-test was used for quan ta ve variables.A p-value of less than 0.05 was considered as sta s cally signifi cant.

Results
Among case group, there were 52 (63.4%) males and 30 (36.5%) females and in the control group there were 54 (65.8%) males and 28 (34.1%)females.Mean birth weight was 2.90±0.32 and 2.90±0.31 in case and control group respec vely.Fi y fi ve (67.0%) neonates were delivered by spontaneous vaginal delivery while 27 (32.9%)were delivered by caesarean sec on in cases group and in control group 51 (62.1%) born through spontaneous vaginal delivery, while 31 (39.0%)neonates were delivered by caesarean sec on.In the present study 59 neonates were found to have NRBC level ≥10/100WBC, out of which 58 (70.7%) were cases and 1(1.2%) was a control.The number of neonates with NRBC levels ≥10/100WBC was signifi cantly more in cases when compared to controls with p-value <0.001, as shown in Table 1.
While comparing the correla on between NRBCs and Apgar score it was found that NRBCs count of ≥10/100WBC were seen more in the newborn who had low 5 min Apgar score and this diff erence was sta s cally signifi cant (p<0.001), as shown in Table 2.
The cut-off NRBC value of ≥10/100WBC has 8.67% sensi vity with a specifi city of 97.75%.NRBC has a posi ve predic ve value of 96.12% with a nega ve predic ve value of 84.47%, as shown in Table 4 In the present study signifi cance and sensi ve area for ROC curve is 0.875 which is sta s cally signifi cant p-value = <0.001.The ROC curve calculated with cut-off NRBC value of ≥10/100WBC.

Discussion
Newborn with perinatal asphyxia adjust to maintain adequate oxygen supply to central organs, aerobic metabolism is supplemented by anaerobic metabolism of glucose and glycogen to maintain cell and organ func on and this anaerobic metabolism produces lac c acid and releases hydrogen ions, which leads to fall in pH, rise in the base defi cit of the extracellular fl uid and glucose store will be reduced in this process.The hypoxic event also induces a compensatory response in the form of exaggerated erythropoesis, resul ng in the release of immature red blood cells (NRBCs) into the fetal circula on.
In the present study 59 neonates were found to have NRBC level ≥10/100WBC, out of which 58 (70.7%) were cases and 1 (1.2%) was a control.The median NRBCs count was 12 and 2.5 for cases and control groups respec vely.The number of neonates with NRBC count of ≥10/100WBC was signifi cantly more in cases when compared to control group (p-value = <0.001).Similar observa on was no ced by Tungalaget al 12 the mean NRBCs count was 11.36±10.7 in case group and 4.83±3.01 in control group, these fi nding were very similar to our study.According to the other studies, the range of NRBC counts was 7.56 to 8.55 in normal term newborns 13,14 .However Phelanet al found the mean NRBCs of 3.4 per 100 WBCs in nonasphyxiated neonates and very high value of 34.5 in asphyxiated neonates 15,16 .Hence it shows that NRBC count increases in the asphyxiated newborn compare to non asphyxiated newborn We had also seen associa on of NRBCs counts with the severity of disease.We found that newborn with NRBCs counts of ≥10/100WBC was associated with low Apgar score and diff erent grades of HIE.In the present study mild HIE, moderate HIE and severe HIE were 10 (16.9%), 29 (49.2%) and 18 (30.5%)respec vely.It indicates high NRBCs count is strongly associated with hypoxic ischaemic encephalopathy and it was sta s cally also signifi cant in our study (p-value = <0.001).This associa on was also compared with other studies, according to Tungalag 12 and Saracoglu et al 13 , there was a signifi cant rela onship between NRBC count and the degree of encephalopathy and this rela onship was also sta s cally signifi cant (p<0.001) 12,13.Manjusa G et al 17 also evaluated the rela onship between HIE staging and nucleated RBC and they found that higher the HIE staging, higher the mean NRBC/100 WBC count.In their study severe HIE was 39.9%, moderate HIE was 45.2% and mild HIE was 18.2%, which is very similar to our study 17 .Neonates diagnosed with HIE were found to have higher NRBC counts, when compared with normal infants.NRBC count is signifi cantly related to the grading of encephalopathy.Some other authors also evaluated the rela on between the severity of asphyxia and cord NRBC count and the rela onship was found to be signifi cant 8,16,18 .
To determine the signifi cance of men oned NRBC level in perinatal asphyxia can be seen from the results of ROC curve test.In the present study signifi cance and sensi ve area for ROC curve is 0.875 which is sta s cally signifi cant with p-value = <0.001and it is very similar with the study done by Tungalaget al 12 , in which they found ROC area = 0.75 with p-value of <0.001.
In our study the cut-off NRBC value of ≥10/100WBC had sensi vity of 70.30%, specifi city of 98.78%, posi ve predic ve value of 98.31% and nega ve predic ve value of 77.14%.According to Manjusha G et al 17 study NRBCs count had a sensi vity of 94.0%, specifi city of 98.0%, posi ve predic ve value 98.0% and nega ve predic ve value of 98.0%.In contrast Hassan B et al 19 found NRBCs count had a sensi vity of 81.3% and specifi city of 94.4%, which were very similar to our study.So it has been found that this simple test can be helpful in the rapid assessment of perinatal asphyxia.

Conclusion
NRBC counts helps to diff eren ate asphyxiated from non-asphyxiated neonates.Neonates with NRBC levels ≥10/100WBC was signifi cantly more in cases when compared to controls with p-value <0.001.The rela onship of NRBCs and the severity of HIE was also found to be sta s cally signifi cant (p-value <0.001).Similarly NRBCs count of ≥10/100WBC were seen more in the newborn who had low 5 min Apgar score and this diff erence was also sta s cally signifi cant (p-value <0.001).Hence the cord blood NRBCs/100 WBCs has a poten al of being used as an early marker for determining the severity and predic ng the outcome of perinatal asphyxia.In resource poor se ngs these bedside diagnos c tests are having high specifi city and sensi vity with low cost and good feasibility.Hence, these markers can be very useful to diff eren ate HIE newborns from non-HIE newborns which will help in appropriate management and be er outcome of these newborns.

Fig 1 :
Fig 1: Nucleated red blood cells as seen in the peripheral smear of cord blood

Fig 2 :
Fig 2: Shows comparison of receiver operator characteris cs (ROC) curves of NRBC

Table 1 :
Nucleated RBC in asphyxiated and non-

Table 2 :
The associa on between Nucleated RBC with Apgar score at 5 min *Chi square test, S = signifi cant

Table 3 :
The associa on between NRBC and severity of HIE *chi square, S = signifi cant

Table 4 :
Sensi vity, specifi city and predic ve values of NRBC