The WHO defines birth asphyxia as failure to initiate and sustain breathing at birth. The National Neonatal-Perinatal Database (NNPD) defines perinatal asphyxia as Apgar score of <7 at 1 min of life. NNPD also defines moderate asphyxia as slow gasping breathing or Apgar score of 4–6 and severe asphyxia as no breathing or Apgar score of 0–3 at 1 min of life.
Birth asphyxia can alter biophysical characteristics of erythrocytes, leukocytes and platelets. Hematological adaption in fetus include long and short term mechanism. Polycythemia is developed during chronic hypoxemia, which leads to the development of hyperviscosity and then causes an increased risk of central nervous system thrombosis. Acute hypoxia involves short-term responses like reduction of heart rate and body temperature, redistribution of circulation, and exaggerated erythropoiesis.
Nucleated red blood cell (NRBC) in the umbilical venous blood of neonates has been reported as a possible marker of perinatal asphyxia. Although the megakaryocytes appear to not be injured by hypoxia, the cells in the bone marrow surrounding them are affected and decrease the release of platelet-promoting factors. Increased destruction of platelets contributes importantly to role in thrombocytopenia of birth asphyxia.
Most of the diagnostic and prognostic parameters used are available in a few selected tertiary care hospitals, are expensive, and require sophisticated equipment, thus rendering them unreachable for most of the population. This problem is further compounded in country like India, where there is a wide gap between the need and accessibility of health services. Therefore, there is a need for simple tests to identify perinatal asphyxia.
Hematological parameters chosen for the study were NRBCs, platelet count, total leukocyte count, hemoglobin, and hematocrit. The purpose of this study was to evaluate the various hematological changes following birth asphyxia Primary objective was to estimate hematological parameters in birth asphyxia in term appropriate for gestational age (AGA) baby. Secondary objectives were to evaluate the correlation between hematological parameters with the severity of birth asphyxia and in the prediction of birth asphyxia outcome.
Materials and Methods
The present study was hospital-based comparative observation study conducted in neonatal units of tertiary hospitals in the northern part of India from May 2019 to May 2020. All term AGA newborns with birth asphyxia admitted in the neonatal unit of our hospital were taken as cases, and healthy newborns from postnatal wards with the same weight and gestation were taken as controls. This study was done after taking ethical committee approval. The ethical committee approval number is 698/MC/EC/2020/dated October 09, 2020.
Cases were defined as mild, moderate, and severe birth asphyxia as criteria put down by NNPD. Inclusion criteria for cases were all term newborns who need resuscitation on admission, with meconium aspiration (nonvigorous), with a history of delayed cry and for controls were healthy term newborns without any need for resuscitation, with no distress, and with no congenital anomaly. Exclusion criteria were prematurity, postmaturity, very low birth weight, small for gestational age, congenital malformation, leaking Per vaginum (PV) ≥24 h, maternal anemia, diabetes, smoking, utero-placental insufficiency, and preeclampsia.
The sample size was calculated at a 95% confidence level and alpha error of 0.05, assuming a standard deviation of 6.39 in the mean value of NRBC among the cases with birth asphyxia and control without birth asphyxia as per the reference article. To detect a difference of at least 3 in the mean value of NRBC among the cases and controls at the study power of 80% required a sample size of 80 participants in each group. Calculation were made using the formula:-Sample size = 4PQ/L2, P = Prevalence, Q = 1– prevalence, L = Error.
All statistical analysis was performed using EPI info 18.104.22.168,CDC,.Atlanta, Georgia (US). Nominal/categorical variables were summarized as frequency and percentage and analyzed using the Chi-square test. Continuous variables were summarized as mean, median, and standard deviation and were analyzed using an independent Student’s t-test. The receiver operating characteristic curve (ROC) graph plotted for NRBC, hemoglobin, platelet, total leukocyte count, hematocrit and then area under curve (AUC) was computed for each index. The correlation between the Apgar score and NRBCs was determined by the Pearson correlation coefficient. A P < 0.05 was taken as statistically significant.
Eighty newborns admitted in NICU on day 1 in neonatal wards who full filled the criteria were taken as cases (Group 1). Eighty newborns (1st postnatal day) of postnatal wards full filling the selection criteria were taken as controls (Group 2). After the approval from the institutional ethics committee and written consent from parents/guardians, detailed antenatal history, natal history, and general physical, neurological, and systemic examination, Sarnat and Sarnat staging of neonates was done, and data were entered into structured pro forma devised by the investigator and validated by senior faculty who have vast working experience in neonatology. Apgar scoring was done prospectively by one of the investigators who were present in labor room/Operation theater (OT) at the time of delivery. Clinical, complete neurological examination and hypoxic-ischemic encephalopathy (HIE) staging was done on days 1 and 3. The correlation was done with hematological parameters and HIE staging to assess the severity and compare outcomes in the form of death or discharge among cases.
Two millimeter cord blood sample was taken for parameters used in the study (NRBC, platelet count, total leukocyte count, hemoglobin, and hematocrit) in an Ethylenediamine tetra acetic acid (EDTA) vial (lavender color vial) immediately after the birth of the baby and was sent to the laboratory immediately. Delayed cord clamping is being practiced at our institute as routine policy, but in our study cases, only cord milking was done as babies required resuscitation at birth. Samples were stored in the refrigerator if there was any delay in processing. Blood counts were determined using a fully automated analyzer (Sysmex Transasia XT 1800i). Using the Coulter principle, cells are sized and counted by detecting and measuring changes in the electrical resistance when a particle passes through a small aperture. Peripheral blood film was done for NRBCs. Blood smear was stained by Leishman stain and observed under microscopy ×100 oil immersion field.
There was no statistically significant difference in relation to sex distribution, birth weight, parity of mothers, and mode of delivery between cases and controls. Weight of the cases and controls was between 2.5 kg and 3.4 kg and with full-term gestation. Both cases and controls were normally distributed. Hence, cases and controls were similar in terms of demographical profile [Table 1].
Among the cases, 42.5% of the babies had severe asphyxia with 1 min Apgar score of 0–3 and 57.5% had moderate asphyxia with Apgar score of 4–6. Fifty-three cases (66.2%) had 5 min Apgar score of 6 or less, of which 5 (6.25%) had severe asphyxia with 5 min Apgar score of 3 or less. Among controls, all babies had 1 min and 5 min Apgar scores of more than 6. Majority of the cases had stage 2 HIE (43.7%) [Table 1].
Hematological investigations, including NRBC, hemoglobin, hematocrit, total leukocyte count, and platelet count, were compared between the two study groups. In the present study, statistically significant higher values were seen in total leukocyte count (P < 0.001), hemoglobin (P < 0.00 1), hematocrit (P = 0.021), and NRBC (P < 0.001) of babies with perinatal asphyxia. Both study groups were similar in terms of platelet count [Table 2].
NRBC and total leukocyte count were statistically positively correlated with the severity of HIE, whereas hemoglobin, hematocrit, and platelet counts were not statistically significant [Table 3].
The mean NRBC count was 30.95 in stage 3 and 21.20 in stage 2 compared to 13.79 in stage 1. NRBC count was increased with decreasing Apgar score at 1 min and 5 min, which was statistically significant (P = 0.0005). Among the cases, a statistically significant negative correlation exists between NRBC count and Apgar score at 1 and 5 min. No such correlation was found among the control.
Receiver operating characteristic (ROC) graphs were plotted to determine cutoff values of different hematological parameters for predicting perinatal asphyxia. ROC graph of NRBC, area under the curve was 0.812; NRBC count cutoff of >20 has sensitivity 83% and specificity 72% [Figure 1]. AUC was 0.654 in the total leukocyte count (TLC) graph, cutoff value of >17.5 × 103/dl TLC has a sensitivity of 75% and specificity of 58.8%, whereas other parameters such as hemoglobin >14.45 gm/dl, hematocrit >45.6%, and platelet count <1.42 × 106 had low sensitivity and specificity values in predicting perinatal asphyxia.
There were a total of 12 deaths in the study. All the deaths occurred in cases with an NRBC count of 20 or more. 46% of total cases with NRBC count of 20 or more died.
The markers of acute asphyxia, like lactate levels, base deficit, and pH, do not determine the chronicity of the disturbances, as it is one of the important antecedents of permanent neurological damage. In contrast, the elevation of NRBC after hypoxia reflects both chronicity and severity of disturbances and may be an important early predictor of poor neurological outcomes.
In the present study, there were 80 babies in both study groups. The demographic details of the mother and term neonates included in the study, like parity, sex of the baby, and mode of delivery, were comparable between both groups. Meena etal. conducted a study on 50 asphyxiated babies, the mean birth weight among cases and control was comparable with our results. A study done by Ganta etal. reported statistically significant higher mean birth weight, whereas Shrivastava etal. reported lower in cases and control, respectively.[9,10] Their study included both preterm and term babies with all birth weight, whereas our study included only term AGA babies.
Findings from other studies revealed that there was no statistically significant difference between the study groups in terms of parity, gender of baby, and mode of delivery which were well comparable with the present study.[8,10,11]
In a study conducted by Shrivastava etal., Apgar at 1 min was 4.17 and 9.3 among cases and controls, respectively, and at 5 min, 6.68 and 9.92 among cases and control, respectively, which were similar to our results.
NRBC are commonly seen in the cord blood of healthy newborns at birth. The number of NRBC is variable but is rarely higher than 10/100 white blood cells (WBC) in healthy term newborns at birth. In the present study, the mean NRBC count in the normal term newborns, i.e., in the control group, was 3.28 ± 2.28 NRBC/100WBC, which is well comparable with other national and international studies done by various authors in the recent past.[9,12,13]
The mean NRBC count in the present study in asphyxiated babies was 21.54 ± 15.36 NRBC/100 WBC, which was significantly higher than normal babies (P < 0.001). The mechanism causing the rapid release of NRBC following perinatal asphyxia is not known, although increased erythropoietin (EPO) results from hypoxia and probably has a major role in the process.[14,15] NRBCs are immature erythrocytes whose production is thought to be driven primarily by the interplay of hypoxia and EPO synthesis. It is possible that increased NRBC production in the immediate neonatal state primarily reflects hypoxic injury. Several studies conducted by different Indian authors have reported an increased NRBC in neonatal blood following perinatal asphyxia.[8,13,18]
In the present study, authors evaluated the relationship between NRBCs and HIE staging and found a positive correlation in cases. The mean NRBC count was 30.95 ± 17.60 in stage 3 and 21.20 ± 15.66 in stage 2 compared to 13.79 ± 5.89 in stage 1. Neonates diagnosed with HIE were found to have higher NRBC counts when compared with control infants. NRBC count was significantly related to the Sarnat’s grading of encephalopathy and also elevated in infants who subsequently died when compared to those who survived. The findings in the present study are consistent with previous studies, and a significantly higher NRBC/100 WBC was found in those who developed HIE in the early neonatal period.[6,9,11,13,19,20]
In the present study, investigators also evaluated the relationship between NRBCs and Apgar scores at 1 and 5 min. They found NRBC of 3.80 ± 3.09, 19.61 ± 16.64, and 21.24 ± 12.24 in the Apgar score range from 7 to 10, 6–4, and 0–3 at 1 min, respectively. At 5 min Apgar score, we found 16.30 ± 6.35, 23.23 ± 15.88, and 33.60 ± 32.11 NRBC/100WBC in Apgar score range from 7 to 10, 6–4, and 0–3, respectively. Similar studies done by Prabhavathi etal. and Ganta etal. also found a significant negative correlation between Apgar score and NRBC at 1 min and at 5 min, which was in accordance with present study.[9,13]
In the present study, authors also evaluated NRBCs in surviving and expired birth asphyxia newborns and observed significantly higher values in the latter. The findings of the study are well comparable with a study conducted by Goel etal. among 50 asphyxiated newborns; 21 (41%) expired and all deaths were associated with a mean NRBC count of 43.19/100 WBC.
According to previous research and the present study, an increasing trend in TLC with the severity of birth asphyxia and worsening grade of HIE was observed. High TLC may be because of oxidative stress and the release of immature leucocytes into the peripheral circulation.[6,8,20]
Other studies conducted by Pol etal. and Koreti etal. found decreasing hemoglobin and hematocrit in term asphyxiated newborns compared to term normal newborns in contrast to present study observations. This may be caused by the intensification of oxidative stress during the prenatal and direct postnatal period, as well as blood redistribution or by hemorrhage.[6,18] The rate of hemoglobin synthesis and of RBC production fall dramatically after birth and remain low for the first 2 weeks of life, probably in response to the sudden increase in tissue oxygenation at birth. The physiological rise in red cell production begins several weeks later.
In the present research work, investigators did not find any correlation between platelet count and birth asphyxia, similar to Koreti etal. study.
In a study conducted by Meena etal., the sensitivity and specificity for NRBC count cutoff of 10 were 66% and 90%, respectively. Other study done in the central part of India by Koreti etal. reported area under the curve for NRBC 0.795 and NRBC count cutoff of ≥10 had a sensitivity of 84.4% and specificity of 64.7% in predicting perinatal asphyxia, similar to present research study.
Limitations of the study
- Study was done in only term newborns with a small number of subjects. Therefore it cannot be generalized to the whole neonatal population
- Study followed babies only up to discharge. Therefore, the association of hematological parameters with neurological outcomes could not be determined
- Study did not correlate NRBC count with the PH, which is reported to be a reliable marker of perinatal asphyxia
- Although we excluded confounding factors like intrauterine growth restriction, diabetes, and preeclampsia from our study, still the complexity of interaction with variables other than asphyxia can affect the blood parameters.
Strength of the study
The study was simple, easily available, and reliable, and can be used as a prognostic marker of birth asphyxia.
NRBC has a significant positive correlation with the severity of hypoxic-ischemic encephalopathy, followed by total leukocyte count and negative correlation with Apgar score. There was a statistically significant difference in the mean value of NRBC, hemoglobin, hematocrit, and total leukocyte count among cases and controls. NRBCs for values more than or equal to 20 can be used as a prognostic marker for assessing the severity and outcome of birth asphyxia.
- Cord blood NRBCs can be used as an effective tool to predict the severity of asphyxia along with other predictors. It also predicts the severity of HIE based on Sarnat and Sarnat staging
- It is a simple, cost-effective, and least invasive test that provides valuable information about the asphyxiated neonates at the earliest so that early treatment can be initiated.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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