Neonatal Nucleated Red Blood Cell Counts

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1 Article Neonatal Nucleated Red Blood Cell Counts Relationship to Abnormal Fetoplacental Circulation Detected by Doppler Studies Roland Axt-Fliedner, MD, Kubilay Ertan, MD, Hans-Joachim Hendrik, MD, Werner Schmidt, MD, PhD Abbreviations NRBC, nucleated red blood cell; S/D, systolic-to-diastolic ratio; SGA, small for gestational age; WBC, white blood cell Received September 26, 2000, from the Department of Obstetrics and Gynecology, University of the Saarland, Homburg/Saar, Germany. Revised manuscript accepted for publication November 22, Address correspondence and reprint requests to Roland Axt- Fliedner, MD, Department of Obstetrics and Gynecology, University of the Saarland, D Homburg/Saar, Germany. Increased neonatal nucleated red blood cell counts are thought to be related to intrauterine hypoxemia. We sought to determine the effect of increasing circulatory impairment in fetuses on the neonatal nucleated red blood cell count. One hundred thirty-four singleton pregnancies were included in the study and were allocated to 4 study groups according to Doppler findings. The systolic-to-diastolic ratios of the umbilical artery, fetal aorta, middle cerebral artery, and uterine arteries were recorded. Fetuses were assigned to the following groups on the basis of the last Doppler examination before delivery: group 1, normal systolic-to-diastolic ratios in the examined vessels; group 2, a systolic-to-diastolic ratio greater than 2 SD above the mean for gestational age in the umbilical artery or fetal aorta and no abnormal Doppler findings in the uterine arteries; group 3, systolic-to-diastolic ratios greater than 2 SD above the mean for gestational age in all examined vessels; and group 4, absence of end-diastolic velocity in the umbilical artery or fetal aorta and systolic-todiastolic ratios greater than 2 SD above the mean for gestational age in the uterine arteries. A blood sample from the umbilical artery was obtained within 1 minute after birth, and nucleated red blood cells per 100 white blood cells were counted by light microscopy. Nucleated red blood cell counts were higher in fetuses in group 4 (median, 72.0; range, 9 720; P <.001) and group 3 (median, 38.4; range, 7 201; P <.001) than in fetuses in group 1 (median, 5.1; range, 0 20). Neonates in group 4 had significantly lower birth weights (P <.001), lower arterial and venous ph values (P <.05), and lower Apgar scores after 5 minutes (P <.01) as well as an increased likelihood of cesarean delivery because of fetal distress (P <.001). The number of fetuses in group 4 with a cord blood base deficit of less than 8 mmol/l was increased. Nucleated red blood cell counts were comparable in fetuses in group 2 (median, 5.4; range, 0 37) and group 1. In groups 1 to 3 no brain-sparing effect occurred, whereas in 15 of 21 cases in group 4 a brain-sparing effect was present. Multivariate analysis revealed that Doppler results of the umbilical artery, fetal aorta, and uterine arteries were independent determinants of neonatal nucleated red blood cell count. Increasing abnormalities seen on fetoplacental Doppler studies are associated with increasing numbers of nucleated red blood cells at birth. Given the known relationship between abnormal Doppler flow and intrauterine hypoxemia, the neonatal nucleated red blood cell count might become an additional valuable tool in the surveillance of growth-restricted fetuses. Key words: nucleated red blood cells; fetus; Doppler ultrasonography by the American Institute of Ultrasound in Medicine J Ultrasound Med 20: , /01/$3.50

2 Neonatal Nucleated Red Blood Cell Counts There is increasing evidence that hypoxicischemic injury of the neonate might not be related to labor but might occur during pregnancy. For example, clinical or biochemical indicators of severe fetal asphyxia are found in only about 10% to 20% of cerebral palsy cases. Therefore, assessment of additional markers of antenatal intrauterine hypoxemia is a relevant task and has important medicolegal aspects. 1 4 Surveillance of fetuses at risk and detection of circulatory deterioration are domains of Doppler ultrasonography. There is evidence that abnormal Doppler findings in the fetal umbilical artery are associated with adverse perinatal outcomes. 5 8 In more recent studies an association between abnormal uterine artery Doppler findings and poor pregnancy outcome was proposed. 9,10 Fetal adaptation to severe intrauterine hypoxemia is known to be associated with an absence of or reversal of flow in the fetal aorta or umbilical artery and centralization of the fetal circulation. 11,12 Furthermore, as an end point of pathologic analysis, abnormal venous flow velocity waveforms have been described as being associated with intrauterine hypoxemia and acidemia. 13 Nucleated red blood cells (NRBCs) are immature erythrocytes frequently seen in variable numbers in the blood of neonates. Generally up to 8 NRBCs per 100 white blood cells (WBCs) are found in the circulating blood of healthy, fullterm neonates. 14,15 Their presence was noted by Anderson in Red blood cell production depends on the stimulation of erythropoietic stem cells by erythropoietin. Because erythropoietin does not cross the placenta, elevated erythropoietin concentrations in fetal cord blood in cases of hypoxemia are of fetal origin. Maternal diabetes, prematurity, chorioamnionitis, intrauterine growth restriction, preeclampsia, and Rh sensitization are thought to be related to increased neonatal NRBC counts. 14,16 Recently, elevated numbers of NRBCs have been reported to be associated with intrauterine hypoxemia and subsequent neurologic impairment. 17,18 To our knowledge there have been few reports on the relationship between abnormal fetal arterial and venous Doppler findings and elevated NRBCs at birth. 13,19 The purpose of this study was to determine whether the extent of circulatory impairment is related to an increase in neonatal NRBC counts. Materials and Methods This study was undertaken in the Department of Obstetrics and Prenatal Ultrasound at the University Hospital of the Saarland after informed consent was obtained from all women. A total of 134 fetuses were included in the study; all had a complete NRBC count and Doppler examination of both uterine arteries, the umbilical artery, the middle cerebral artery, and the fetal aorta within 5 days (mean, 2.7 days) before delivery. The patients were recruited from the population of pregnant women who were referred to our ultrasonography department for evaluation of fetal well-being because of suspected fetal growth restriction, oligohydramnios, maternal hypertension, preeclampsia, or pathologic fetal heart rate patterns. Multiple pregnancies, chorioamnionitis, maternal renal disease, maternal diabetes, maternal cardiovascular disease other than hypertension, and fetuses with chromosomal or structural anomalies were excluded. The Doppler examinations were performed in the second and third trimesters with Acuson (Mountain View, CA) 128 XP/10 or Siemens (Erlangen, Germany) Sonoline-Elegra ultrasonography equipment. Doppler data from both uterine arteries, the umbilical artery, the fetal aorta, and the middle cerebral artery were obtained. A 3.5- or 5-MHz curved array transducer with a high-pass filter of 100 Hz was used. The Doppler examination was performed with the patient lying in a semirecumbent position. For uterine artery Doppler imaging the transducer was placed in the right or left lower part of the abdomen. Color Doppler imaging was used to localize the main uterine artery superior to the crossing of the external iliac artery. Pulsed Doppler imaging was then used to obtain flow velocity waveforms. The examination was repeated on the opposite side. Doppler waveforms were obtained from a free-floating central part of the umbilical artery in the absence of body movements, fetal breathing, or cardiac arrhythmia with the sample volume covering the whole vessel. The fetal aorta was localized in its abdominal part at the origin of the renal arteries. The middle cerebral artery was visualized at about 1 cm from its origin in the circle of Willis in an axial view. Care was taken to minimize fetal head compression, because this is known to influence the flow velocity waveforms of the middle cerebral arteries. For every vessel exam- 184 J Ultrasound Med 20: , 2001

3 Axt-Fliedner et al ined, 5 consecutive waveforms of similar quality were accepted for analysis. The ratio of mean peak systolic to diastolic velocity (S/D ratio) was determined. Abnormal umbilical, uterine, and fetal aorta Doppler results were those greater than 2 SD above the mean for gestational age of our local reference ranges. 20 Uterine notching was registered, but in view of the relatively small study group, we did not examine uterine notching separately. Fetal brain sparing was presumed when the S/D ratio was less than 2 SD below the mean of our local reference ranges for the middle cerebral artery. Immediately after delivery a blood sample was drawn from the umbilical artery and vein by needle aspiration. The NRBC count was assessed from the umbilical artery sample. Two blood smears of each sample stained with Wright s stain were prepared, and the number of NRBCs per 100 WBCs was assessed by light microscopy by 2 trained observers who were blinded to the study groups. 15 On the basis of the Doppler findings, fetuses were allocated to 4 groups (Table 1). Group 1 (n = 54) had normal Doppler findings; group 2 (n = 39) had abnormal Doppler findings confined to the umbilical artery or fetal aorta; group 3 (n = 20) had abnormal Doppler findings in the umbilical artery, the fetal aorta, and both uterine arteries. Group 4 (n = 21) had no presence of end-diastolic velocity in the umbilical artery or fetal aorta. The estimated time of delivery was assessed by the last menstrual data and a vaginal ultrasonographic measurement of crown-rump length within the first 12 weeks after the last menstruation. Small for gestational age (SGA) was defined as a fetal abdominal circumference smaller than that of the fifth percentile for gestational age in our reference ranges. 21 According to Davey and McGillivray 22 previously normotensive women with diastolic blood pressure of 140/90 mm Hg or higher measured on 2 occasions 6 hours apart after 20 weeks of gestation and with proteinuria of at least 1+ on protein stick testing or a proteinuria level of greater than 0.3 g/24 h were considered preeclamptic. 22 Histologic chorioamnionitis was diagnosed on the basis of the presence of acute inflammatory cell infiltration of both layers of the membranes. Prematurity was defined as delivery before 37 completed weeks of gestation. Outcome measures included NRBC counts, birth weight, gestational age at delivery, mode of delivery (vaginal delivery or cesarean delivery because of clinical signs of fetal distress [e.g., abnormal fetal heart rate patterns, meconiumstained amniotic fluid, and umbilical acidosis]), arterial and venous ph, arterial base excess, Apgar score, admission to the neonatal intensive care unit, and length of stay in the neonatal intensive care unit. Results were analyzed with SPSS statistical software (SPSS Inc, Chicago, IL). Distribution of NRBCs was nonnormal. Neonatal NRBC counts are presented as median and range; other results are reported as mean ± SD. Statistical analysis included the Mann-Whitney U test, Student t test, χ 2 analysis of variance, and stepwise regression analysis. Bonferroni correction for multiple testing was used. P <.05 was considered significant. Results A total of 134 fetuses were included in the study. All of the women examined were white. Maternal characteristics concerning age, parity, and pregnancy complications did not show significant differences. Table 1 shows the 4 study groups with respect to the Doppler findings. Fifty-four fetuses (40.2%) had normal Doppler findings in the umbilical artery, and 93 fetuses (69.4%) had normal Doppler flow velocity waveforms in both uterine arteries. In 38 cases (28.3%) we found abnormal Doppler results in both uterine arteries, and in 20 cases (14.9%) we found pathologic S/D ratios in the umbilical artery and fetal aorta. Fifteen (11.1%) of 134 fetuses had brain-sparing effects, and 21 (15.6%) had no end-diastolic velocity in the umbilical artery or fetal aorta. These cases were allocated to group 4. Table 1. Doppler Results in the Different Study Groups Umbilical Artery Uterine No. of Group or Fetal Aorta Arteries Cases 1 Normal Normal 54 2 Abnormal Normal 39 3 Both abnormal Both abnormal 20 4 Absence of end- Both abnormal 21 diastolic velocity (18/21) (brain-sparing effect, 15/21) Abnormal was defined as greater than 2 SD above locally used reference ranges. 20 J Ultrasound Med 20: ,

4 Neonatal Nucleated Red Blood Cell Counts Table 2. Delivery Outcomes Table 2 summarizes the obstetric data of the 4 study groups. Fifty fetuses (37.3%) were SGA. In 10 cases (7.5%) we found preeclampsia alone. In 20 cases (14.9%) we found SGA fetuses and preeclampsia (Table 2). As expected, fetuses in group 4 had the highest prematurity rate (100% versus 14.8% in group 1; P <.001) and the lowest rate of vaginal delivery (0% versus 55.5% in group 1; P <.001). Fetuses in group 4 were more likely to be delivered by cesarean section because of fetal distress (90.4% versus 3.7% in group 1; P <.001). The number of neonatal intensive care unit admissions increased progressively among the groups, with the highest number in group 4 (100% versus 7.4% in group 1; P <.001). Perinatal data are given in Table 3. Nucleated red blood cell counts showed a stepwise increase from group 1 (median, 5.1; range, 0 20) to group 4 (median, 72.0; range, 9 720). The highest NRBC counts were observed in group 4 (P <.001 versus group 1; P <.05 versus group 3). NRBC counts were comparable for fetuses in group 2 (median, 5.4; range, 0 37) and group 1 (median, 5.1; range, 0 20). Gestational age at delivery decreased in study groups 3 and 4 (group 1: median, 38.3 weeks; range, weeks; group 4: median, 30.4 weeks; range, weeks), and birth weights were progressively lower among the 4 groups (group 1: median, 3423 g; range, g; group 4: median, 900 g; range, g; P <.001). All data examined with respect to blood gases showed significant differences in group 4, except the base excess values, which were close to demonstrating statistical significance (P =.05 versus group 1). Arterial and venous ph values Outcome Group 1 Group 2 Group 3 Group 4 (n = 54) (n = 39) (n = 20) (n = 21) SGA 12 (22.2) 11 (28.2) 6 (30.0) 21 (100)* Preeclampsia 3 (5.5) 2 (5.1) 5 (25.0) 0 Both (45.0) 11 (52.3) Preterm delivery 8 (14.8) 5 (12.8) 19 (95) 21 (100)* Vaginal delivery 30 (55.5) 29 (74.3) 4 (20.0) 0* Cesarean delivery 24 (44.5) 10 (25.7) 16 (80.0) 21 (100)* Cesarean delivery for fetal distress 2 (3.7) 3 (7.6) 14 (70.0)* 19 (90.4)* NICU admission 4 (7.4) 8 (20.5) 18 (90.0)* 21 (100)* Neonatal death (9.5) Data are presented as numbers with percentages in parentheses. NICU indicates neonatal intensive care unit. *P <.001 versus group 1; P <.01 versus group 3; P <.01 versus group 1; P <.05 versus group 1; P <.05 versus group 3. were significantly lower in group 4 than in group 1 (P <.05 versus group 1), and the different Apgar scores were lower in groups 3 and 4 than in group 1. The 2 neonatal deaths occurred in group 4 on the 5th and 15th days of life because of multiorgan failure. In both cases high NRBC counts were observed at birth (203 and 273 NRBCs per 100 WBCs, respectively). On the 3rd day of life the NRBC count was still more than 5 NRBCs per 100 WBCs. Stepwise regression was conducted with the neonatal NRBC count as the dependent variable and birth weight, gestational age at delivery, and Doppler indices as dependent variables. This analysis demonstrated that birth weight (P <.001; r 2 = 0.17), gestational age at delivery (P <.001; r 2 = 0.30), Doppler results in the uterine arteries (P <.001; r 2 = 0.14), and Doppler results in the fetal vessels examined (P <.001; r 2 = 0.17) in groups 3 and 4 were significant independent determinants of the neonatal NRBC count at birth. Finally, there was no correlation between the neonatal NRBC count and arterial and venous ph values or between the neonatal NRBC count and arterial base excess values. However, we detected a weak correlation between 1-, 5-, and 10-min Apgar scores and the NRBC count (P =.05). Discussion Modern perinatal medicine has contributed to an important reduction of perinatal mortality over the last 3 decades, from about 3% to 0.5% to 0.6% according to a perinatal database in Germany. 23 Conversely, a comparable reduction of perinatal neurologic morbidity was not observed. In Germany about 1000 children per year are still born with a cerebral hypoxicischemic injury, and bilateral cerebral palsy is found in 1 in 1000 live births. 23 For many years cerebral hypoxic-ischemic injury of the neonate was attributed to intrapartum asphyxia. During the past several years, however, it has become evident that long-term neurologic impairment of the neonate might not be due primarily to intrapartum asphyxia but, rather, might occur long before the fetal life stage. In addition, in only 10% to 20% of cases of cerebral palsy has an association with clinical or biochemical markers of fetal asphyxia been observed J Ultrasound Med 20: , 2001

5 Axt-Fliedner et al Table 3. Perinatal Outcomes Outcome Group 1 Group 2 Group 3 Group 4 NRBCs/100 WBCs 5.1 (0 20) 5.4 (0 37) 38.4* (7 201) 72.0* (9 720) Gestational age at delivery, wk 38.3 ( ) 39.0 ( ) 32.6* ( ) 30.4* ( ) Birth weight, g 3423 ( ) 3011 ( ) 1848* ( ) 900* ( ) Apgar score at 1 min 8.4 (4 10) 8.7 (6 10) 6.6 (1 10) 6.0* (3 8) Apgar score at 5 min 9.3 (6 10) 9.6 (7 10) 8.0 (1 10) 8.0 (1 10) Apgar score at 10 min 9.8 (8 10) 9.5 (8 10) 8.2 (3 10) 9.0 (6 10) Apgar score <7 at 1 min, n (%) 7 (13) 7 (18) 10 (50) 13 (62) Apgar score <7 at 5 min, n (%) 1 (1.9) 4 (10) 3 (15) 1 (4.8) Arterial ph 7.29 ( ) 7.28 ( ) 7.25 ( ) 7.24 ( ) Venous ph 7.35 ( ) 7.34 ( ) 7.30 ( ) 7.27 ( ) Base excess, mmol/l 3.4 (1.7 to 14.5) 3.9 ( 0.7 to 11.0) 4.1 (0.6 to 14.0) 4.2 ( 3.7 to 17.3) Arterial ph <7.20, n (%) 3 (6) 8 (21) 3 (20) 3 (14) Base excess < 8 mmol/l, n (%) 2 (4) 3 (8) 2 (10) 4 (19) Data are presented as medians with ranges in parentheses or, as indicated under Outcome, as numbers with percentages in parentheses. *P <.001 versus group 1; P <.05 versus group 3; P <.01 versus group 3; P <.01 versus group 1; P <.05 versus group 1. A major challenge of modern perinatal medicine in the future will be to reduce longterm neurologic impairment of the neonate. Therefore, the search for additional markers of antenatal chronic fetal hypoxemia is an important and relevant task. 24 Fetal erythropoiesis is mainly driven by the hypoxia-erythropoietin-nrbc precursors. 25 The number of NRBCs circulating in the blood of a healthy, full-term infant is variable but normally does not exceed 8 or 9 NRBCs per 100 WBCs. 14 In a previous study we reported comparable NRBC counts in healthy, full-term infants and slightly higher counts in post-term infants. 15 In previous studies, elevated NRBC counts have been proposed as a marker of intrauterine hypoxemia in full-term infants. Korst et al 18 reported distinct NRBC patterns in relation to the timing of fetal injury in neurologically impaired neonates. They found higher NRBC counts in cases of suspected injury before admission to the hospital than in cases of injury occurring acutely during birth. Korst et al 18 concluded that NRBC levels may assist in determining the timing of fetal neurologic injury. Phelan et al 17 found in their series of 46 singleton, full-term, neurologically impaired neonates that the number of NRBCs was lower when fetal asphyxia occurred closer to birth. Therefore, the number of NRBCs at birth should aid in determining the time of fetal asphyxia. In an animal model, release of reticulocytes after hypoxia was not seen until the second or third day after the hypoxic stimulus. 26 Given this observation, elevated NRBC counts should be found in the cord blood of the neonate only if the hypoxic insult occurs before the onset of labor. Therefore, elevated NRBC counts after acute intrapartum hypoxia are not expected in postpartum cord blood but might occur during the neonatal period. This is in accordance with our previous work, which did not show any relation between mode or duration of delivery and NRBCs in uncomplicated full- and post-term pregnancies. 15 The association of increased erythropoietin levels in fetal plasma with intrauterine growth restriction has been reported previously. 27 Furthermore, the link between poor fetal oxygenation and acidemia with elevated resistance indices in fetal arteries examined by Doppler ultrasonography has been assessed by invasive methods. Elevated NRBC counts associated with higher resistance indices in fetal vessels have been previously documented by Groenenberg et al. 28 Recently, this finding was confirmed by Bernstein et al 19 and Baschat et al. 13 We sought to determine in a large series whether circulatory deterioration in appropriate-for-gestational age and SGA fetuses detected by Doppler ultrasonography is associated with increased NRBC counts. The results of the current study indicate that NRBC counts are increased when a higher degree of abnormality is seen on Doppler imaging of the fetoplacental unit. The absence of end-diastolic flow velocity in the umbilical artery or fetal aorta was the strongest independent determinant of elevated NRBC counts at birth. Abnormal J Ultrasound Med 20: ,

6 Neonatal Nucleated Red Blood Cell Counts Doppler findings in the umbilical artery or fetal aorta alone did not result in increased NRBC counts at birth. Our results are in accordance with previous studies that investigated the relationship between NRBC counts and abnormal Doppler findings. Bernstein and coworkers 19 compared NRBC counts obtained postpartum in SGA fetuses (n = 52) with end-diastolic velocity present (n = 33) and in SGA fetuses with absent or reversed end-diastolic velocity (n = 19). They found a significant increase of NRBCs in those fetuses with absent or reversed end-diastolic velocity. 19 More recently, Baschat and coworkers 13 found increased NRBCs in intrauterine growth-restricted fetuses (n = 84) to be associated with abnormal arterial and venous Doppler flow results. They reported higher numbers of NRBCs in fetuses with centralization compared with fetuses with only high resistance indices in the umbilical artery, and they documented the highest NRBC counts in fetuses with abnormal flow velocity waveforms in the inferior vena cava or the ductus venosus. Nicolini and coworkers 29 found a similar association between abnormal umbilical artery Doppler results and NRBC counts in venous cord blood obtained by cordocentesis in 46 growth-restricted fetuses. Not unexpectedly, the number of growthrestricted fetuses was highest in group 4, probably because of pathologic fetoplacental gas exchange and poor nutrient transport across the placenta. We also observed preeclampsia in about 50% of the cases in group 4. The prematurity rate in group 4 was 100%. Therefore, the cesarean delivery rate was high in this group, mainly because of the diagnosis of fetal distress, and all neonates in this group had to be transferred to the neonatal intensive care unit. When we analyzed cord blood gas results, we found significantly lower arterial and venous ph values in the group with the highest degree of abnormal Doppler findings; arterial and venous ph values progressively decreased as the degree of abnormal Doppler findings increased. We observed the same relationship between the arterial base excess values and the degree of circulatory deterioration, but this association did not reach statistical significance (P =.05). Centralization of the fetal circulation and the absence of end-diastolic velocities in the umbilical artery are known to be related to low arterial cord blood oxygen content, low ph values, and increased lactate concentrations in cord blood. 11,12 Thus, our results confirm deterioration of fetoplacental gas exchange in cases of increasing Doppler abnormalities, which would then lead to stimulation of the erythropoietin axis and subsequently higher counts of NRBC precursors in fetal blood, as found in this study. Interestingly, Baschat and coworkers 13 did not find significantly reduced arterial PO 2 levels among groups with high umbilical resistance index values, centralization of the fetal circulation, and abnormal venous index values, although they found increased NRBC counts. This might indicate that arterial PO 2 does not correctly indicate tissue oxygenation or tissue hypoxemia. On the other hand, other studies have found high NRBC counts in the absence of subsequent reticulocytosis or within 2 hours after acute fetal hypoxemia. These results propose alternative pathways of stimulation of fetal erythropoiesis. 30,31 One limitation of the present study might be that NRBC counts were assessed after both vaginal and cesarean delivery in groups 1 to 3. However, we previously reported similar NRBC counts in full- and post-term fetuses delivered either vaginally or by cesarean section; thus, the method of delivery might not have a significant effect on NRBC counts. 15 In conclusion, we have shown a relationship between elevated neonatal NRBC counts and increasing Doppler abnormalities in the fetoplacental unit. Perinatal data were worst in those fetuses with an absence of end-diastolic velocity in the umbilical artery or fetal aorta and the highest NRBC counts. Thus, we speculate that intrauterine hypoxemia occurs in fetuses with severe Doppler abnormalities, as shown by the higher NRBC counts and lower arterial ph values. Given the known relationship between abnormal Doppler flow and intrauterine hypoxemia, the determination of NRBCs in cord blood might become an additional valuable tool in the diagnosis of intrauterine hypoxemia. Studies to further elucidate the relationship among erythropoietin concentration, lactate concentration, and NRBC counts in cord blood of compromised fetuses are under way in our institution. 188 J Ultrasound Med 20: , 2001

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