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Nucleated red blood cell count at birth as an index of perinatal brain damage
Nucleated red blood cell count at birth as an index of perinatal brain damage Giuseppe Buonocore, MD,a Serafina Perrone, MD,a Dino Gioia,a Maria Gabriella Gatti, MD,a Cosimo Massafra, MD,b Rosaria Agosta, MD,a and Rodolfo Bracci, MDa Siena, Italy OBJECTIVE: The prognostic value of the nucleated red blood cell count at birth with respect to perinatal brain damage and neonatal outcome was assessed in infants at high risk of having neurologic damage. STUDY DESIGN: The nucleated red blood cell count at birth, pulsed Doppler ultrasonography in the cerebral arteries, cranial fontanelle sonograms, and neurodevelopmental status were evaluated in 337 newborn infants. RESULTS The nucleated red blood cell count at birth was significantly higher (1) in neonates with abnormal Doppler ultrasonographic parameters for the cerebral arteries at 48 to 72 hours after birth than in healthy neonates, (2) in 6-month-old infants with sequelae of hypoxic-ischemic encephalopathy than in healthy infants, and (3) in 3-year-old children with abnormal developmental status than in those with no abnormalities at follow-up. Significant correlations were observed between the nucleated red blood cell count and gestational age, Apgar score at 1 and 5 minutes, pH, base deficit, fraction of inspired oxygen, blood oxygen content, and birth weight. CONCLUSIONS: The nucleated red blood cell count at birth not only reflects a response of the infant to perinatal hypoxia but is also a reliable index of perinatal brain damage. (Am J Obstet Gynecol 1999;181:1500-5.)
Key words: Newborn infant, erythroblasts, hypoxia, neurology, developmental disability
Perinatal hypoxia may cause cell necrosis or delayed neuronal death in the newborn infant.1 Therefore fetal hypoxia can have an important influence on survival and quality of life and requires careful follow-up.2 Several indexes such as changes in fetal heart rate (FHR) patterns, ultrasonographic and pulse Doppler findings, and meconium-stained fluid have been suggested as markers of intrauterine fetal distress, but an exact evaluation of perinatal brain damage is still difficult to establish.3-6 It was recently reported that the nucleated red blood cell count indicates fetal hypoxia in term and preterm newborn infants.7, 8 The only known stimulus of erythropoietin and nucleated red blood cell generation is tissue hypoxia, which was well documented in human fetal and animal models.9-14 Distinct nucleated red blood cell patterns seem to be related to the timing of fetal injury, and this suggests that nucleated red blood cell counts may assist
From the Institute of Preventive Paediatrics and Neonatologya and the Institute of Obstetrics and Gynaecology,b University of Siena. Supported by the Italian Ministry for University and ScientificTechnologic Research (MURST Funds 40% and 60%). Received for publication January 28, 1999; revised April 5, 1999; accepted June 9, 1999. Reprint requests: Giuseppe Buonocore, MD, Institute of Preventive Paediatrics and Neonatology, University of Siena, Policlinico “Le Scotte,” V. le Bracci No. 36, 53100 Siena, Italy. Copyright © 1999 by Mosby, Inc. 0002-9378/99 $8.00 + 0 6/1/100658
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in determining the timing of fetal neurologic impairment.8-15 However, correlations of the nucleated red blood cell count with follow-up data on neurodevelopmental status have not previously been made. The aim of this study was to compare nucleated red blood cell counts with the common markers of hypoxia and to assess the prognostic value of the nucleated red blood cell count in the first hours after birth with respect to hypoxic perinatal brain damage and neonatal outcome in a large cohort of neonates. Methods Patients. Three hundred thirty-seven newborn infants were the subjects of the present study (for clinical characteristics see Table I). Two hundred eighty-two infants were consecutively admitted to the neonatal intensive care unit at the University of Siena Institute of Preventive Paediatrics and Neonatology during the period from June 1, 1991–May 30, 1995. Using a random numbers table, we selected 55 healthy term newborn infants from the nursery, 1 or 2 babies every month during the study period, as control subjects. All babies not born in the clinic and all babies with congenital malformation, an inborn error of metabolism, blood group incompatibility, sepsis, a mother with diabetes, multiple gestation, or anemia were excluded from the study to eliminate risks that could affect the number of circulating erythroblasts. The entire study was strictly masked.
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Table I. Characteristics of patients Extremely preterm (n = 47) Gestational age (wk, mean ± SD and range) Gender (male/female ratio) Birth weight (kg, mean ± SD and range) Small for gestational age (No.) Apgar score (mean ± SD and range) At 1 min At 5 min Cardiotocography (No.) Normal Bradycardia Late intrapartum decelerations of FHR Loss of irregularity Blood gas analysis (mean ± SD and range) pH Base deficit (mmol/L) Perinatal hypoxia (No.)*
25.57 ± 1.12 (24-27) 22/25 0.81 ± 0.18 (0.51-1.22) 6 (12.7%)
Preterm (n = 185) 32.46 ± 2.45 (28-36) 91/94 1.79 ± 0.60 (0.72-3.43) 31 (16.7%)
Term (n = 105) 38.78 ± 1.29 (37-41) 56/49 3.04 ± 0.67 (1.30-4.95) 9 (8.6%)
3.3 ± 2.0 (1-8) 6.0 ± 2.1 (1-10)
4.9 ± 2.6 (1-10) 7.8 ± 1.8 (1-10)
6.3 ± 3.1 (0-10) 8.4 ± 2.1 (1-10)
23 2 2 1
107 9 22 18
76 5 17 6
7.18 ± 0.14 (6.82-7.41) 8.5 ± 5.8 (1.1-20.3) 42
7.24 ± 0.11 (6.88-7.48) 6.1 ± 5.5 (3.6-28.6) 107
7.28 ± 0.14 (6.94-7.45) 5.4 ± 6.1 (3.3-25.6) 50
*Criteria for perinatal hypoxia consisted of the following: pH, ≤7.20 in the umbilical vein; Apgar score, <7 at 5 minutes; and fraction of inspired oxygen needed for resuscitation immediately after delivery, ≥0.4.
Thirty subjects died in the first 3 months after birth as a consequence of perinatal abnormalities (pulmonary hemorrhage, persistent pulmonary hypertension, severe intracranial hemorrhage, central venous thrombosis), ascertained by autopsy. They were matched with babies of the same birth weight and gestational age, randomly selected from the group of survivors, to exclude any effects of gestational age and birth weight on nucleated red blood cell count. The babies were divided into 3 groups according to gestational age. Gestational age was determined from the last menstrual period and early ultrasonography; if there was any discrepancy, neonatal Dubowitz evaluation was used.16 Babies were classified as small for gestational age if their birth weight was >2 SD below the mean, according to the growth chart of Yudkin et al.17 One hundred twenty-eight babies (39 extremely preterm, 47 preterm, 42 term) were born by vaginal delivery; 98 babies (56 preterm and 42 term) were born by elective cesarean delivery; 111 babies (8 extremely preterm, 82 preterm, 21 term) were born by emergency cesarean delivery. One hundred five newborn infants (34 extremely preterm, 57 preterm, 14 term) needed assisted ventilation at birth. Pulsed Doppler sonograms of the anterior and middle cerebral arteries were obtained for all babies at 48 to 72 hours after birth. The peak systolic, peak end-systolic, peak end-diastolic, and time-average mean velocities, together with the Pourcelot resistance index, were determined. Brain Doppler patterns regarded as abnormal included reduction in flow velocities with retrograde flow during early diastole and changes in the Pourcelot index. The reduction in flow velocities was considered abnormal when systolic flow velocity was ≤0.20 m/s, Pourcelot index was ≤0.5, and diastolic reverse flow was present. Cranial fontanelle sonograms, with standard sagittal and
coronal cuts, were obtained for all babies at birth and again 6 months later. Ultrasonographic findings regarded as abnormal included sequelae of hypoxic-ischemic encephalopathy (focal or multifocal cortical necrosis with cystic encephalomalacia), 15%; periventricular leukomalacia, 80%; and cerebral cortical atrophy, 5%. Neonatal scans and pulsed Doppler findings were evaluated by a single neonatologist experienced in ultrasonography. The diagnosis of hypoxic encephalopathy was confirmed by computed tomography or magnetic resonance imaging. Mean nucleated red blood cell counts at birth were compared in babies with normal and in those with abnormal brain Doppler and ultrasonographic findings. Because gestational age and birth weight are independent risk factors for intraventricular hemorrhage and periventricular leukomalacia and because the nucleated red blood cell count is known to be inversely related to gestational age and birth weight, we checked for these confounding variables in the following way. The nucleated red blood cell count of each neonate with abnormal ultrasonographic and Doppler findings was compared with that of a healthy neonate matched for gestational age (±1.5 weeks) and birth weight (±10%). Neurodevelopmental status was assessed, with the investigator masked to the perinatal nucleated red blood cell count, on the third day after birth and repeated at age 1, 2, 4, 12, 18, 24, and 36 months, according to the criteria of Allen and Capute.18 Twenty-three babies with minor or major disabilities were classified as having abnormalities. They were matched with babies of the same birth weight and gestational age, randomly selected from the children with normal neurodevelopmental status, to exclude any effects of gestational age and birth weight on the nucleated red blood cell count. Neonates were considered hypoxic according to the
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B
Fig 1. Nucleated red blood cell counts at birth in babies with normal and abnormal findings on brain pulsed-Doppler sonograms at 48 to 72 hours after birth (A) and in 6-month-old babies with normal and abnormal findings on brain sonograms (B). Open bars, Normal findings; filled bars, abnormal findings; asterisk, P = .0001; double dagger, P = .009.
following criteria: pH ≤7.20 in the umbilical vein, Apgar score ≤6 at 5 minutes, and fraction of inspired oxygen needed for resuscitation immediately after delivery ≥0.4. These criteria, less severe than the current definition from The American College of Obstetricians and Gynecologists,19 were chosen to evaluate the prognostic value of the nucleated red blood cell count even in babies with mild hypoxia. The study was approved by the University of Siena Medical Faculty Human Ethics Committee. Methods. Heparinized blood samples were obtained from the umbilical vein, after cord clamping, immediately after delivery. A complete blood cell count was performed, and the total white blood cell count was initially determined in the laboratory of our neonatal intensive care unit. The nucleated red blood cell count was expressed as the absolute erythroblast count (nucleated red blood cells per cubic millimeter), calculated by light microscopic examination of May-Grünwald-Giemsa–stained blood smears. Amniotic fluid was examined for meconium by amnioscopy, 2 hours before delivery. Cardiotocography was carried out in the peripartal period with a HewlettPackard 8040A (Hewlett-Packard, Andover, Mass) recorder. This recorder was calibrated at every analysis to have comparable analyses. Blood gas analysis was measured with a model ABL 505 analyzer (Radiometer, Copenhagen, Denmark) immediately after blood sampling. Blood oxygen content was calculated as follows: Hemoglobin (g/dL) × oxygen (1.36 mL/g hemoglobin) × Oxygen saturation of hemoglobin (%). Brain pulsed Doppler ultrasonography and brain ultrasonography were performed 48 to 72 hours after birth and repeated at age 2 weeks to check for intraventricular hemorrhage and periventricular leukomalacia with a 7.5-MHz probe hav-
Fig 2. Nucleated red blood cell count at birth in 3-year-old children with normal and abnormal neurodevelopmental status, according to criteria of Allen and Capute.18 Open bar, Normal findings; filled bar, abnormal findings; asterisk, P = .001.
ing a transmitted pulsed-Doppler frequency of 7.5 to 5 MHz (Interspec Apogee, Milan, Italy). The anterior cerebral artery was found by color Doppler ultrasonography, and the gain, volume sample, and velocity scale were adjusted to achieve the best recording of the arterial signal. The data, expressed as mean ± SD, were analyzed for statistically significant differences by the 2-tailed Student t test for unpaired data and by linear correlation, with the SPSS/PC +4 (SPSS Inc, Chicago, Ill) statistical package. Results Mean nucleated red blood cell counts at birth were significantly higher in extremely preterm (8520.72 ± 1620.11) than in preterm (4548.07 ± 473.69) and term (1689.35 ± 290.06) babies (P = .002 and P = .0001, respectively). In extremely preterm babies (group 1), no differences in nucleated red blood cell counts at birth were observed between babies delivered vaginally and those delivered by cesarean and between babies who underwent intubation and those who did not. In preterm and term babies (group 2 and group 3, respectively), the nucleated red blood cell count was found to be significantly higher (1) in neonates with cardiotocographic evidence of an abnormal FHR pattern (loss of baseline heart rate frequency, late intrapartum decelerations of the fetal heart rate, bradycardia, or a combination of these) than in those with a normal FHR pattern, (2) in babies born by emergency cesarean delivery than in those born by elective cesarean or vaginal delivery, and (3) in babies with than in those without hypoxia (Table II). Statistically significant differences in nucleated red blood cell counts at birth were found (1) between extremely preterm infants who were small and those who were appropriately sized for gestational age (20935.33 ±
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Table II. Nucleated red blood cell count in relation to cardiotocography, mode of delivery, and perinatal hypoxia in preterm and term newborn infants
Variable Cardiotocography Normal findings Bradycardia Late intrapartum decelerations of FHR Loss of irregularity Delivery Vaginal Elective cesarean Emergency cesarean Perinatal hypoxia* Babies with hypoxia Healthy babies
Nucleated red blood cell count in preterm group (No./mm3, mean ± SD)
No. (N = 185)
Statistical significance
Nucleated red blood cell count in term group No. (No./mm3, mean ±SD) (N = 105)
Statistical significance
2077.74 ± 272.40 6843.22 ± 2520.45 9164.95 ± 1894.71
107 9 22
P = .0001 P = .0001 P = .0001
792.10 ± 129.30 2925.60 ± 882.82 4506.62 ± 1335.93
76 5 17
P = .0001 P = .0001 P = .0001
7759.72 ± 1879.65
18
P = .0001
7611.00 ± 4718.05
6
P = .0001
3479.38 ± 702.03 2929.57 ± 875.38 6265.93 ± 753.39
47 56 82
P = .01 P = .005 P = .01, P = .005
1843.71 ± 780.21 1088.42 ± 204.44 5335.33 ± 1647.05
42 42 21
P = .03 P = .001 P = .03, P = .001
6602.61 ± 740.64 1729.66 ± 239.75
107 78
P = .0001 P = .0001
4252.88 ± 927.40 409.96 ± 98.97
50 55
P = .0001 P = .0001
*Criteria for perinatal hypoxia: pH, ≤7.20 in the umbilical vein; Apgar score, <7 at 5 minutes; and fraction of inspired oxygen needed for resuscitation immediately after delivery, ≥0.4.
6994.11 vs 6703.95 ± 1385.87; P = .002); (2) between preterm infants who were small and those who were appropriately sized for gestational age (7728.64 ± 1414.04 vs 3907.83 ± 478.81; P = .002); (3) between term newborn infants with meconium-stained amniotic fluid and those with clear fluid (4304.40 ± 1882.04 vs 1666.23 ± 347.44; P = .03); (4) between term newborn infants who underwent intubation and those who did not (3145.69 ± 462.03 vs 409.96 ± 98.97; P = .003); and (5) between babies who died and the survivor control group (10479.63 ± 1780.32 vs 2622.46 ± 579.26; P = .0001). The nucleated red blood cell count at birth was significantly higher (1) in neonates with an abnormal cerebral artery Doppler ultrasonographic picture at 48 to 72 hours after birth than in those with normal patterns (Fig 1); (2) in 6-month-old infants with ultrasonographic evidence of hypoxic-ischemic encephalopathy than in babies of the same age without such evidence (Fig 1); (3) in 3-year-old children with abnormal developmental status than in children of the same age with no abnormalities (Fig 2). Significant correlations were observed in the whole infant population between the nucleated red blood cell count at birth and (1) gestational age (r = –0.31; P = .0001; n = 337); (2) birth weight (r = –0.28; P = .0001; n = 337); (3) Apgar score at 1 minute (r = –0.29; P = .0001; n = 337); (4) Apgar score at 5 minutes: (r = –0.32; P = .0001; n = 337); (5) pH (r = –0.32; P = .0001; n = 337); (6) base deficit (r = –0.30; P = .0001; n = 335); (7) fraction of inspired oxygen needed for resuscitation immediately after delivery (r = 0.38; P = .0001; n = 326); and (8) blood oxygen content (r = –0.43; P = .0001; n = 328). Comment Many conditions are known to induce intrauterine hypoxia and to generate a large number of circulating nu-
cleated red blood cells.20 The number of nucleated red blood cells is variable and inversely related to gestational age and birth weight on the first day after birth.7, 15 Our results in term and preterm neonates are in keeping with these observations. The increase observed in nucleated red blood cell counts associated with fetal growth restriction is also in line with previous reports of significantly higher nucleated red blood cell counts in small-for-gestational-age than in adequate-for-gestational-age infants, even when matched for gestational age.7, 21 The differences found between term newborn infants with meconium-stained amniotic fluid and those with clear amniotic fluid support the previously reported hypothesis that intrauterine asphyxia may cause release of meconium and result in an adverse outcome.22 The high nucleated red blood cell count found in babies born with meconium-stained amniotic fluid could be explained by increased erythropoietin levels, as previously reported in these babies.4 Changes in FHR patterns, which have been regarded as the principal diagnostic criterion for fetal hypoxia (monitoring of FHR patterns was therefore recently proposed as obligatory during delivery), are a poor predictor of fetal acidosis and of outcome for babies.3, 23 However, we found a significantly higher nucleated red blood cell count in term and preterm babies with anomalous FHR patterns than in those with a normal pattern, and we also found a significant correlation between nucleated red blood cell counts and outcome for infants. These findings suggest that accurate FHR monitoring may provide information on basic fetal status and may predict hypoxia that could develop during labor. In fact, an increase in nucleated red blood cells may be the consequence of fetal tissue hypoxia before or during labor. This suggestion is further supported by the fact that the
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highest nucleated red blood cell counts were observed in pregnancies ending in emergency cesarean delivery because of a nonreassuring FHR tracing. Higher nucleated red blood cell counts were observed in neonates who underwent intubation than in those who did not, and counts were higher in babies with hypoxia than in those without hypoxia. Highly significant correlations between nucleated red blood cell counts and pH, base deficit, fraction of inspired oxygen immediately after delivery, and Apgar scores at 1 and 5 minutes were found in all infant populations. This finding strongly suggests that hypoxia and perinatal distress are the 2 main factors responsible for increasing nucleated red blood cell counts, probably by stimulating hyperproduction of erythropoietin. Most evidence in human subjects suggests that the time interval between the erythropoietin rise and the peak nucleated red blood cell count is at least 24 to 48 hours and declines after 7 days.7 This observation has been documented in both human fetal and animal models.10, 11 Recently, Leikin et al7 failed to observe a correlation between nucleated red blood cell counts and the presence or absence of intraventricular hemorrhage and periventricular leukomalacia in the first day after birth. This lack of correlation led the authors to suggest that the nucleated red blood cell count plays no part in determining the timing of neurologic injury. On the other hand, when Green et al24 studied appropriately sized babies with or without intraventricular hemorrhage who were born at ≤32 weeks, they found higher nucleated red blood cell counts in the first 6 days after birth in infants in whom severe intraventricular hemorrhage developed than in control subjects. The authors concluded that elevated or increasing nucleated red blood cell counts in a preterm newborn infant could be a marker of impending or already severe intraventricular hemorrhage. Our results are in keeping with these observations, with higher nucleated red blood cell counts in neonates with an abnormal Doppler ultrasonographic pattern than in those with a normal pattern, which suggests that the nucleated red blood cell count may be of predictive value. The contrasting results can be explained by the method chosen to evaluate brain damage. We used the Pourcelot index instead of ultrasonography because the Pourcelot index may be more sensitive than the real-time image for documenting brain damage caused by asphyxia.25, 26 The outcome at 6 months included a significantly higher nucleated red blood cell count in infants with ultrasonographic evidence of sequelae of hypoxic-ischemic encephalopathy than in babies with normal patterns. These findings are in line with the reports of Korst et al8 and Phelan et al,15 who observed higher nucleated red blood cell counts in cord blood of neonates with neurologic impairment. Their data were obtained by retrospective review. Our prospective study stresses the nucle-
December 1999 Am J Obstet Gynecol
ated red blood cell count at birth as a reliable index of perinatal hypoxia. Moreover, higher nucleated red blood cell counts in the first hours after birth in neurologically impaired children, in comparison with normal children on follow-up evaluation after 3 years, may be considered a marker of poor neurodevelopmental outcome. That the babies who died had the highest nucleated red blood cell counts is consistent with this hypothesis. In conclusion, our results indicate that the nucleated red blood cell count is helpful in identifying perinatal hypoxia and in predicting neurodevelopmental outcome. Although the timing of perinatal hypoxic brain damage may vary and is often unknown, detection of an elevated number of nucleated red blood cells during the neonatal period in children with neurodevelopmental impairment, reliably diagnosed at follow-up at the age of 3 years, suggests that the nucleated red blood cell count not only reflects an adaptive response of the infant to perinatal distress but also is predictive of an increased risk of brain damage. REFERENCES
1. Fellman V, Raivio KO. Reperfusion injury as the mechanism of brain damage after perinatal asphyxia. Pediatr Res 1997;41:599606. 2. Maier RF, Gunther A, Vogel M, Dudenhausen JW, Obladen M. Umbilical venous erythropoietin and umbilical arterial pH in relation to morphologic placental abnormalities. Obstet Gynecol 1994;84:81-7. 3. Saling E. Comments on past and present situation of intensive monitoring of the fetus during labor. J Perinat Med 1996;24:713. 4. Richey SD, Ramin S, Bawdon RE, Roberts SW, Dak J, Roberts J, et al. Markers of acute and chronic asphyxia in infants with meconium-stained amniotic fluid. Am J Obstet Gynecol 1995;172:1212-5. 5. Ruth V, Autti-Ramo I, Granstrom ML, Korkman M, Raivio KO. Prediction of perinatal brain damage by cord plasma vasopressin, erythropoietin, and hypoxanthine values. J Pediatr 1988;113:880-5. 6. Thilaganathan B, Athanasiou S, Ozmen S, Creighton S, Watson NR, Nicolaides KH. Umbilical cord blood erythroblast count as an index of intrauterine hypoxia. Arch Dis Child 1994;70:F192-4. 7. Leikin E, Verma U, Klein S, Tejani N. Relationship between neonatal nucleated red blood cell counts and hypoxic-ischemic injury. Obstet Gynecol 1996;87:439-43. 8. Korst LM, Phelan JP, Ahn MO, Martin GI. Nucleated red blood cells: an update on the marker for fetal asphyxia. Am J Obstet Gynecol 1996;176:843-6. 9. Buonocore G, Vezzosi P, Perrone S, Gioia D, Bracci R. Erythroblast count in newborn infants in relation to different markers of fetal hypoxia [abstract]. Pediatr Res 1996;40:522A. 10. Maier RF, Bohme K, Dudenhausen JW, Obladen M. Cord blood erythropoietin in relation to different markers of fetal hypoxia. Obstet Gynecol 1993;81:575-80. 11. Eckhard K-U, Hartmann W, Vetter J, Pohlandt F, Burghardt R, Kurtz A. Serum immunoreactive erythropoietin of children in health and disease. Eur J Pediatr 1990;149:459-64. 12. Ruth V, Fyhrquist F, Clemons G, Raivio KO. Cord plasma vasopressin, erythropoietin, and hypoxanthine are indices of asphyxia at birth. Pediatr Res 1988;24:490-4. 13. Clemons GK, Fitzsimmons SL, DeManincor D. Immunoreactive erythropoietin concentrations in fetal and neonatal rats and the effects of hypoxia. Blood 1986;68:892-9. 14. Widness JA, Teramo KA, Clemons GK, Garcia JF, Cavalieri RL, Piasecki GJ, et al. Temporal response of immunoreactive ery-
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thropoietin to acute hypoxemia in fetal sheep. Pediatr Res 1986;20:15-9. Phelan JP, Ahn MO, Korst LM, Martin GI. Nucleated red blood cells: a marker for fetal asphyxia? Am J Obstet Gynecol 1995;173:1380-4. Dubowitz LMS, Dubowitz V, Goldberg CG. Clinical assessment of the newborn infant. J Pediatr 1970;77:1-10. Yudkin PL, Aboualfa M, Eyre JA, Redman CW, Wilkinson AR. New birthweight and head circumference centiles for gestational age 24 to 42 weeks. Early Hum Dev 1987;15:45-52. Allen MC, Capute AJ. Neonatal neurodevelopmental examination as a predictor of neuromotor outcome in premature infants. Pediatrics 1989;83:498-506. The American College of Obstetricians and Gynecologists. Fetal distress and birth asphyxia. Washington: The College; 1994. Committee Opinion No.: 137. Smith CA, Nelson NM. The physiology of the newborn infant. Springfield (IL): Charles C Thomas; 1976. p. 269.
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21. Philip AGS, Tito AM. NRBC counts in small for gestational age infants with very low birth weight. Am J Dis Child 1989;143: 164-9. 22. Wiswell TE, Bent RC. Meconium staining and the meconium aspiration syndrome. Pediatr Clin North Am 1993;40: 955-81. 23. Wolff F. Obligatory monitoring methods during delivery. Z Geburtshilfe Neonatol 1997;201:1-5. 24. Green DW, Hendon B, Mimouni FB. Nucleated erythrocytes and intraventricular hemorrhage in preterm neonates. Pediatrics 1995;96:475-8. 25. Gonzalez de Dios J, Moyra M, Izura V. Variations in cerebral blood flow in various states of severe neonatal hypoxic-ischemic encephalopathy. Rev Neurol 1995;23:639-43. 26. Boishardy N, Granry JC, Jacob JP, Houi N, Fournier D, Delhumeau A. Value of transcranial Doppler ultrasonography in the management of severe head injuries. Ann Fr Anesth Reanim 1994;13:172-6.
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Key words: Newborn infant, erythroblasts, hypoxia, neurology, developmental disability
Perinatal hypoxia may cause cell necrosis or delayed neuronal death in the newborn infant.1 Therefore fetal hypoxia can have an important influence on survival and quality of life and requires careful follow-up.2 Several indexes such as changes in fetal heart rate (FHR) patterns, ultrasonographic and pulse Doppler findings, and meconium-stained fluid have been suggested as markers of intrauterine fetal distress, but an exact evaluation of perinatal brain damage is still difficult to establish.3-6 It was recently reported that the nucleated red blood cell count indicates fetal hypoxia in term and preterm newborn infants.7, 8 The only known stimulus of erythropoietin and nucleated red blood cell generation is tissue hypoxia, which was well documented in human fetal and animal models.9-14 Distinct nucleated red blood cell patterns seem to be related to the timing of fetal injury, and this suggests that nucleated red blood cell counts may assist
From the Institute of Preventive Paediatrics and Neonatologya and the Institute of Obstetrics and Gynaecology,b University of Siena. Supported by the Italian Ministry for University and ScientificTechnologic Research (MURST Funds 40% and 60%). Received for publication January 28, 1999; revised April 5, 1999; accepted June 9, 1999. Reprint requests: Giuseppe Buonocore, MD, Institute of Preventive Paediatrics and Neonatology, University of Siena, Policlinico “Le Scotte,” V. le Bracci No. 36, 53100 Siena, Italy. Copyright © 1999 by Mosby, Inc. 0002-9378/99 $8.00 + 0 6/1/100658
1500
in determining the timing of fetal neurologic impairment.8-15 However, correlations of the nucleated red blood cell count with follow-up data on neurodevelopmental status have not previously been made. The aim of this study was to compare nucleated red blood cell counts with the common markers of hypoxia and to assess the prognostic value of the nucleated red blood cell count in the first hours after birth with respect to hypoxic perinatal brain damage and neonatal outcome in a large cohort of neonates. Methods Patients. Three hundred thirty-seven newborn infants were the subjects of the present study (for clinical characteristics see Table I). Two hundred eighty-two infants were consecutively admitted to the neonatal intensive care unit at the University of Siena Institute of Preventive Paediatrics and Neonatology during the period from June 1, 1991–May 30, 1995. Using a random numbers table, we selected 55 healthy term newborn infants from the nursery, 1 or 2 babies every month during the study period, as control subjects. All babies not born in the clinic and all babies with congenital malformation, an inborn error of metabolism, blood group incompatibility, sepsis, a mother with diabetes, multiple gestation, or anemia were excluded from the study to eliminate risks that could affect the number of circulating erythroblasts. The entire study was strictly masked.
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Table I. Characteristics of patients Extremely preterm (n = 47) Gestational age (wk, mean ± SD and range) Gender (male/female ratio) Birth weight (kg, mean ± SD and range) Small for gestational age (No.) Apgar score (mean ± SD and range) At 1 min At 5 min Cardiotocography (No.) Normal Bradycardia Late intrapartum decelerations of FHR Loss of irregularity Blood gas analysis (mean ± SD and range) pH Base deficit (mmol/L) Perinatal hypoxia (No.)*
25.57 ± 1.12 (24-27) 22/25 0.81 ± 0.18 (0.51-1.22) 6 (12.7%)
Preterm (n = 185) 32.46 ± 2.45 (28-36) 91/94 1.79 ± 0.60 (0.72-3.43) 31 (16.7%)
Term (n = 105) 38.78 ± 1.29 (37-41) 56/49 3.04 ± 0.67 (1.30-4.95) 9 (8.6%)
3.3 ± 2.0 (1-8) 6.0 ± 2.1 (1-10)
4.9 ± 2.6 (1-10) 7.8 ± 1.8 (1-10)
6.3 ± 3.1 (0-10) 8.4 ± 2.1 (1-10)
23 2 2 1
107 9 22 18
76 5 17 6
7.18 ± 0.14 (6.82-7.41) 8.5 ± 5.8 (1.1-20.3) 42
7.24 ± 0.11 (6.88-7.48) 6.1 ± 5.5 (3.6-28.6) 107
7.28 ± 0.14 (6.94-7.45) 5.4 ± 6.1 (3.3-25.6) 50
*Criteria for perinatal hypoxia consisted of the following: pH, ≤7.20 in the umbilical vein; Apgar score, <7 at 5 minutes; and fraction of inspired oxygen needed for resuscitation immediately after delivery, ≥0.4.
Thirty subjects died in the first 3 months after birth as a consequence of perinatal abnormalities (pulmonary hemorrhage, persistent pulmonary hypertension, severe intracranial hemorrhage, central venous thrombosis), ascertained by autopsy. They were matched with babies of the same birth weight and gestational age, randomly selected from the group of survivors, to exclude any effects of gestational age and birth weight on nucleated red blood cell count. The babies were divided into 3 groups according to gestational age. Gestational age was determined from the last menstrual period and early ultrasonography; if there was any discrepancy, neonatal Dubowitz evaluation was used.16 Babies were classified as small for gestational age if their birth weight was >2 SD below the mean, according to the growth chart of Yudkin et al.17 One hundred twenty-eight babies (39 extremely preterm, 47 preterm, 42 term) were born by vaginal delivery; 98 babies (56 preterm and 42 term) were born by elective cesarean delivery; 111 babies (8 extremely preterm, 82 preterm, 21 term) were born by emergency cesarean delivery. One hundred five newborn infants (34 extremely preterm, 57 preterm, 14 term) needed assisted ventilation at birth. Pulsed Doppler sonograms of the anterior and middle cerebral arteries were obtained for all babies at 48 to 72 hours after birth. The peak systolic, peak end-systolic, peak end-diastolic, and time-average mean velocities, together with the Pourcelot resistance index, were determined. Brain Doppler patterns regarded as abnormal included reduction in flow velocities with retrograde flow during early diastole and changes in the Pourcelot index. The reduction in flow velocities was considered abnormal when systolic flow velocity was ≤0.20 m/s, Pourcelot index was ≤0.5, and diastolic reverse flow was present. Cranial fontanelle sonograms, with standard sagittal and
coronal cuts, were obtained for all babies at birth and again 6 months later. Ultrasonographic findings regarded as abnormal included sequelae of hypoxic-ischemic encephalopathy (focal or multifocal cortical necrosis with cystic encephalomalacia), 15%; periventricular leukomalacia, 80%; and cerebral cortical atrophy, 5%. Neonatal scans and pulsed Doppler findings were evaluated by a single neonatologist experienced in ultrasonography. The diagnosis of hypoxic encephalopathy was confirmed by computed tomography or magnetic resonance imaging. Mean nucleated red blood cell counts at birth were compared in babies with normal and in those with abnormal brain Doppler and ultrasonographic findings. Because gestational age and birth weight are independent risk factors for intraventricular hemorrhage and periventricular leukomalacia and because the nucleated red blood cell count is known to be inversely related to gestational age and birth weight, we checked for these confounding variables in the following way. The nucleated red blood cell count of each neonate with abnormal ultrasonographic and Doppler findings was compared with that of a healthy neonate matched for gestational age (±1.5 weeks) and birth weight (±10%). Neurodevelopmental status was assessed, with the investigator masked to the perinatal nucleated red blood cell count, on the third day after birth and repeated at age 1, 2, 4, 12, 18, 24, and 36 months, according to the criteria of Allen and Capute.18 Twenty-three babies with minor or major disabilities were classified as having abnormalities. They were matched with babies of the same birth weight and gestational age, randomly selected from the children with normal neurodevelopmental status, to exclude any effects of gestational age and birth weight on the nucleated red blood cell count. Neonates were considered hypoxic according to the
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A
December 1999 Am J Obstet Gynecol
B
Fig 1. Nucleated red blood cell counts at birth in babies with normal and abnormal findings on brain pulsed-Doppler sonograms at 48 to 72 hours after birth (A) and in 6-month-old babies with normal and abnormal findings on brain sonograms (B). Open bars, Normal findings; filled bars, abnormal findings; asterisk, P = .0001; double dagger, P = .009.
following criteria: pH ≤7.20 in the umbilical vein, Apgar score ≤6 at 5 minutes, and fraction of inspired oxygen needed for resuscitation immediately after delivery ≥0.4. These criteria, less severe than the current definition from The American College of Obstetricians and Gynecologists,19 were chosen to evaluate the prognostic value of the nucleated red blood cell count even in babies with mild hypoxia. The study was approved by the University of Siena Medical Faculty Human Ethics Committee. Methods. Heparinized blood samples were obtained from the umbilical vein, after cord clamping, immediately after delivery. A complete blood cell count was performed, and the total white blood cell count was initially determined in the laboratory of our neonatal intensive care unit. The nucleated red blood cell count was expressed as the absolute erythroblast count (nucleated red blood cells per cubic millimeter), calculated by light microscopic examination of May-Grünwald-Giemsa–stained blood smears. Amniotic fluid was examined for meconium by amnioscopy, 2 hours before delivery. Cardiotocography was carried out in the peripartal period with a HewlettPackard 8040A (Hewlett-Packard, Andover, Mass) recorder. This recorder was calibrated at every analysis to have comparable analyses. Blood gas analysis was measured with a model ABL 505 analyzer (Radiometer, Copenhagen, Denmark) immediately after blood sampling. Blood oxygen content was calculated as follows: Hemoglobin (g/dL) × oxygen (1.36 mL/g hemoglobin) × Oxygen saturation of hemoglobin (%). Brain pulsed Doppler ultrasonography and brain ultrasonography were performed 48 to 72 hours after birth and repeated at age 2 weeks to check for intraventricular hemorrhage and periventricular leukomalacia with a 7.5-MHz probe hav-
Fig 2. Nucleated red blood cell count at birth in 3-year-old children with normal and abnormal neurodevelopmental status, according to criteria of Allen and Capute.18 Open bar, Normal findings; filled bar, abnormal findings; asterisk, P = .001.
ing a transmitted pulsed-Doppler frequency of 7.5 to 5 MHz (Interspec Apogee, Milan, Italy). The anterior cerebral artery was found by color Doppler ultrasonography, and the gain, volume sample, and velocity scale were adjusted to achieve the best recording of the arterial signal. The data, expressed as mean ± SD, were analyzed for statistically significant differences by the 2-tailed Student t test for unpaired data and by linear correlation, with the SPSS/PC +4 (SPSS Inc, Chicago, Ill) statistical package. Results Mean nucleated red blood cell counts at birth were significantly higher in extremely preterm (8520.72 ± 1620.11) than in preterm (4548.07 ± 473.69) and term (1689.35 ± 290.06) babies (P = .002 and P = .0001, respectively). In extremely preterm babies (group 1), no differences in nucleated red blood cell counts at birth were observed between babies delivered vaginally and those delivered by cesarean and between babies who underwent intubation and those who did not. In preterm and term babies (group 2 and group 3, respectively), the nucleated red blood cell count was found to be significantly higher (1) in neonates with cardiotocographic evidence of an abnormal FHR pattern (loss of baseline heart rate frequency, late intrapartum decelerations of the fetal heart rate, bradycardia, or a combination of these) than in those with a normal FHR pattern, (2) in babies born by emergency cesarean delivery than in those born by elective cesarean or vaginal delivery, and (3) in babies with than in those without hypoxia (Table II). Statistically significant differences in nucleated red blood cell counts at birth were found (1) between extremely preterm infants who were small and those who were appropriately sized for gestational age (20935.33 ±
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Table II. Nucleated red blood cell count in relation to cardiotocography, mode of delivery, and perinatal hypoxia in preterm and term newborn infants
Variable Cardiotocography Normal findings Bradycardia Late intrapartum decelerations of FHR Loss of irregularity Delivery Vaginal Elective cesarean Emergency cesarean Perinatal hypoxia* Babies with hypoxia Healthy babies
Nucleated red blood cell count in preterm group (No./mm3, mean ± SD)
No. (N = 185)
Statistical significance
Nucleated red blood cell count in term group No. (No./mm3, mean ±SD) (N = 105)
Statistical significance
2077.74 ± 272.40 6843.22 ± 2520.45 9164.95 ± 1894.71
107 9 22
P = .0001 P = .0001 P = .0001
792.10 ± 129.30 2925.60 ± 882.82 4506.62 ± 1335.93
76 5 17
P = .0001 P = .0001 P = .0001
7759.72 ± 1879.65
18
P = .0001
7611.00 ± 4718.05
6
P = .0001
3479.38 ± 702.03 2929.57 ± 875.38 6265.93 ± 753.39
47 56 82
P = .01 P = .005 P = .01, P = .005
1843.71 ± 780.21 1088.42 ± 204.44 5335.33 ± 1647.05
42 42 21
P = .03 P = .001 P = .03, P = .001
6602.61 ± 740.64 1729.66 ± 239.75
107 78
P = .0001 P = .0001
4252.88 ± 927.40 409.96 ± 98.97
50 55
P = .0001 P = .0001
*Criteria for perinatal hypoxia: pH, ≤7.20 in the umbilical vein; Apgar score, <7 at 5 minutes; and fraction of inspired oxygen needed for resuscitation immediately after delivery, ≥0.4.
6994.11 vs 6703.95 ± 1385.87; P = .002); (2) between preterm infants who were small and those who were appropriately sized for gestational age (7728.64 ± 1414.04 vs 3907.83 ± 478.81; P = .002); (3) between term newborn infants with meconium-stained amniotic fluid and those with clear fluid (4304.40 ± 1882.04 vs 1666.23 ± 347.44; P = .03); (4) between term newborn infants who underwent intubation and those who did not (3145.69 ± 462.03 vs 409.96 ± 98.97; P = .003); and (5) between babies who died and the survivor control group (10479.63 ± 1780.32 vs 2622.46 ± 579.26; P = .0001). The nucleated red blood cell count at birth was significantly higher (1) in neonates with an abnormal cerebral artery Doppler ultrasonographic picture at 48 to 72 hours after birth than in those with normal patterns (Fig 1); (2) in 6-month-old infants with ultrasonographic evidence of hypoxic-ischemic encephalopathy than in babies of the same age without such evidence (Fig 1); (3) in 3-year-old children with abnormal developmental status than in children of the same age with no abnormalities (Fig 2). Significant correlations were observed in the whole infant population between the nucleated red blood cell count at birth and (1) gestational age (r = –0.31; P = .0001; n = 337); (2) birth weight (r = –0.28; P = .0001; n = 337); (3) Apgar score at 1 minute (r = –0.29; P = .0001; n = 337); (4) Apgar score at 5 minutes: (r = –0.32; P = .0001; n = 337); (5) pH (r = –0.32; P = .0001; n = 337); (6) base deficit (r = –0.30; P = .0001; n = 335); (7) fraction of inspired oxygen needed for resuscitation immediately after delivery (r = 0.38; P = .0001; n = 326); and (8) blood oxygen content (r = –0.43; P = .0001; n = 328). Comment Many conditions are known to induce intrauterine hypoxia and to generate a large number of circulating nu-
cleated red blood cells.20 The number of nucleated red blood cells is variable and inversely related to gestational age and birth weight on the first day after birth.7, 15 Our results in term and preterm neonates are in keeping with these observations. The increase observed in nucleated red blood cell counts associated with fetal growth restriction is also in line with previous reports of significantly higher nucleated red blood cell counts in small-for-gestational-age than in adequate-for-gestational-age infants, even when matched for gestational age.7, 21 The differences found between term newborn infants with meconium-stained amniotic fluid and those with clear amniotic fluid support the previously reported hypothesis that intrauterine asphyxia may cause release of meconium and result in an adverse outcome.22 The high nucleated red blood cell count found in babies born with meconium-stained amniotic fluid could be explained by increased erythropoietin levels, as previously reported in these babies.4 Changes in FHR patterns, which have been regarded as the principal diagnostic criterion for fetal hypoxia (monitoring of FHR patterns was therefore recently proposed as obligatory during delivery), are a poor predictor of fetal acidosis and of outcome for babies.3, 23 However, we found a significantly higher nucleated red blood cell count in term and preterm babies with anomalous FHR patterns than in those with a normal pattern, and we also found a significant correlation between nucleated red blood cell counts and outcome for infants. These findings suggest that accurate FHR monitoring may provide information on basic fetal status and may predict hypoxia that could develop during labor. In fact, an increase in nucleated red blood cells may be the consequence of fetal tissue hypoxia before or during labor. This suggestion is further supported by the fact that the
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highest nucleated red blood cell counts were observed in pregnancies ending in emergency cesarean delivery because of a nonreassuring FHR tracing. Higher nucleated red blood cell counts were observed in neonates who underwent intubation than in those who did not, and counts were higher in babies with hypoxia than in those without hypoxia. Highly significant correlations between nucleated red blood cell counts and pH, base deficit, fraction of inspired oxygen immediately after delivery, and Apgar scores at 1 and 5 minutes were found in all infant populations. This finding strongly suggests that hypoxia and perinatal distress are the 2 main factors responsible for increasing nucleated red blood cell counts, probably by stimulating hyperproduction of erythropoietin. Most evidence in human subjects suggests that the time interval between the erythropoietin rise and the peak nucleated red blood cell count is at least 24 to 48 hours and declines after 7 days.7 This observation has been documented in both human fetal and animal models.10, 11 Recently, Leikin et al7 failed to observe a correlation between nucleated red blood cell counts and the presence or absence of intraventricular hemorrhage and periventricular leukomalacia in the first day after birth. This lack of correlation led the authors to suggest that the nucleated red blood cell count plays no part in determining the timing of neurologic injury. On the other hand, when Green et al24 studied appropriately sized babies with or without intraventricular hemorrhage who were born at ≤32 weeks, they found higher nucleated red blood cell counts in the first 6 days after birth in infants in whom severe intraventricular hemorrhage developed than in control subjects. The authors concluded that elevated or increasing nucleated red blood cell counts in a preterm newborn infant could be a marker of impending or already severe intraventricular hemorrhage. Our results are in keeping with these observations, with higher nucleated red blood cell counts in neonates with an abnormal Doppler ultrasonographic pattern than in those with a normal pattern, which suggests that the nucleated red blood cell count may be of predictive value. The contrasting results can be explained by the method chosen to evaluate brain damage. We used the Pourcelot index instead of ultrasonography because the Pourcelot index may be more sensitive than the real-time image for documenting brain damage caused by asphyxia.25, 26 The outcome at 6 months included a significantly higher nucleated red blood cell count in infants with ultrasonographic evidence of sequelae of hypoxic-ischemic encephalopathy than in babies with normal patterns. These findings are in line with the reports of Korst et al8 and Phelan et al,15 who observed higher nucleated red blood cell counts in cord blood of neonates with neurologic impairment. Their data were obtained by retrospective review. Our prospective study stresses the nucle-
December 1999 Am J Obstet Gynecol
ated red blood cell count at birth as a reliable index of perinatal hypoxia. Moreover, higher nucleated red blood cell counts in the first hours after birth in neurologically impaired children, in comparison with normal children on follow-up evaluation after 3 years, may be considered a marker of poor neurodevelopmental outcome. That the babies who died had the highest nucleated red blood cell counts is consistent with this hypothesis. In conclusion, our results indicate that the nucleated red blood cell count is helpful in identifying perinatal hypoxia and in predicting neurodevelopmental outcome. Although the timing of perinatal hypoxic brain damage may vary and is often unknown, detection of an elevated number of nucleated red blood cells during the neonatal period in children with neurodevelopmental impairment, reliably diagnosed at follow-up at the age of 3 years, suggests that the nucleated red blood cell count not only reflects an adaptive response of the infant to perinatal distress but also is predictive of an increased risk of brain damage. REFERENCES
1. Fellman V, Raivio KO. Reperfusion injury as the mechanism of brain damage after perinatal asphyxia. Pediatr Res 1997;41:599606. 2. Maier RF, Gunther A, Vogel M, Dudenhausen JW, Obladen M. Umbilical venous erythropoietin and umbilical arterial pH in relation to morphologic placental abnormalities. Obstet Gynecol 1994;84:81-7. 3. Saling E. Comments on past and present situation of intensive monitoring of the fetus during labor. J Perinat Med 1996;24:713. 4. Richey SD, Ramin S, Bawdon RE, Roberts SW, Dak J, Roberts J, et al. Markers of acute and chronic asphyxia in infants with meconium-stained amniotic fluid. Am J Obstet Gynecol 1995;172:1212-5. 5. Ruth V, Autti-Ramo I, Granstrom ML, Korkman M, Raivio KO. Prediction of perinatal brain damage by cord plasma vasopressin, erythropoietin, and hypoxanthine values. J Pediatr 1988;113:880-5. 6. Thilaganathan B, Athanasiou S, Ozmen S, Creighton S, Watson NR, Nicolaides KH. Umbilical cord blood erythroblast count as an index of intrauterine hypoxia. Arch Dis Child 1994;70:F192-4. 7. Leikin E, Verma U, Klein S, Tejani N. Relationship between neonatal nucleated red blood cell counts and hypoxic-ischemic injury. Obstet Gynecol 1996;87:439-43. 8. Korst LM, Phelan JP, Ahn MO, Martin GI. Nucleated red blood cells: an update on the marker for fetal asphyxia. Am J Obstet Gynecol 1996;176:843-6. 9. Buonocore G, Vezzosi P, Perrone S, Gioia D, Bracci R. Erythroblast count in newborn infants in relation to different markers of fetal hypoxia [abstract]. Pediatr Res 1996;40:522A. 10. Maier RF, Bohme K, Dudenhausen JW, Obladen M. Cord blood erythropoietin in relation to different markers of fetal hypoxia. Obstet Gynecol 1993;81:575-80. 11. Eckhard K-U, Hartmann W, Vetter J, Pohlandt F, Burghardt R, Kurtz A. Serum immunoreactive erythropoietin of children in health and disease. Eur J Pediatr 1990;149:459-64. 12. Ruth V, Fyhrquist F, Clemons G, Raivio KO. Cord plasma vasopressin, erythropoietin, and hypoxanthine are indices of asphyxia at birth. Pediatr Res 1988;24:490-4. 13. Clemons GK, Fitzsimmons SL, DeManincor D. Immunoreactive erythropoietin concentrations in fetal and neonatal rats and the effects of hypoxia. Blood 1986;68:892-9. 14. Widness JA, Teramo KA, Clemons GK, Garcia JF, Cavalieri RL, Piasecki GJ, et al. Temporal response of immunoreactive ery-
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21. Philip AGS, Tito AM. NRBC counts in small for gestational age infants with very low birth weight. Am J Dis Child 1989;143: 164-9. 22. Wiswell TE, Bent RC. Meconium staining and the meconium aspiration syndrome. Pediatr Clin North Am 1993;40: 955-81. 23. Wolff F. Obligatory monitoring methods during delivery. Z Geburtshilfe Neonatol 1997;201:1-5. 24. Green DW, Hendon B, Mimouni FB. Nucleated erythrocytes and intraventricular hemorrhage in preterm neonates. Pediatrics 1995;96:475-8. 25. Gonzalez de Dios J, Moyra M, Izura V. Variations in cerebral blood flow in various states of severe neonatal hypoxic-ischemic encephalopathy. Rev Neurol 1995;23:639-43. 26. Boishardy N, Granry JC, Jacob JP, Houi N, Fournier D, Delhumeau A. Value of transcranial Doppler ultrasonography in the management of severe head injuries. Ann Fr Anesth Reanim 1994;13:172-6.
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