Association of ABO incompatibility with elevation of nucleated red blood cell counts in term neonates

Association of ABO incompatibility with elevation of nucleated red blood cell counts in term neonates Kathleen M. Hanlon-Lundberg, MD, and Russell S. Kirby, PhD Milwaukee, Wisconsin OBJECTIVE: Nucleated red blood cells in the circulation in term neonates have been associated with a wide range of pathologic conditions. We sought to examine the relationship between nucleated red blood cells in the circulation of term neonates and maternal-neonatal blood type compatibility. STUDY DESIGN: We prospectively collected umbilical blood from all live-born neonates delivered at our institution. Venous blood was analyzed for nucleated red blood cells and is reported as the number of nucleated red blood cells per 100 white blood cells. We reviewed maternal and neonatal records for neonates born at ≥37 weeks’ gestation for correlative clinical data. Statistical analysis was performed with the SAS statistical software package (version 6.12; SAS Institute, Inc, Cary, NC). Kruskal-Wallis analysis was used as a nonparametric test. RESULTS: We evaluated 1661 neonates delivered during the study period and found a mean (±SD) of 9.29 ± 18.56 nucleated red blood cells per 100 white blood cells (range, 0-327 nucleated red blood cells per 100 white blood cells). Nucleated red blood cell counts were lower in ABO-compatible maternal-fetal dyads (mean ± SD, 8.29 ± 12.84 nucleated red blood cells per 100 white blood cells; range, 0-216 nucleated red blood cells per 100 white blood cells) than in ABO-incompatible dyads (mean ± SD, 13.16 ± 13.16 nucleated red blood cells per 100 white blood cells; range, 0-327 nucleated red blood cells/100 white blood cells; P = .006). Neonates of mothers with blood groups A and B had significantly lower nucleated red blood cell counts (P < .05). Dyads with maternal type O and neonate type B had significantly higher nucleated red blood cell counts (P < .002). Nonparametric testing determined that type O mother and type B neonate combinations had significantly higher umbilical cord nucleated red blood cell counts (P < .001). CONCLUSION: Maternal-fetal ABO incompatibility is associated with elevation of nucleated red blood cell count in term neonates. Nucleated red blood cell elevation does not always connote a serious pathologic process, however, because ABO incompatibility usually does not adversely affect neonatal outcome. The clinical significance of an elevated nucleated red blood cell count thus is limited. (Am J Obstet Gynecol 2000;183:1532-6.)

Key words: ABO incompatibility, hemolytic disease of the neonate, nucleated red blood cells

Nucleated red blood cells are immature red blood cells. The presence of increased numbers of nucleated red blood cells in the circulation of term infants has been associated with diverse pathologic conditions, such as hemolytic disease of the newborn, intrauterine growth restriction, maternal diabetes mellitus, preeclampsia, and perinatal brain damage.1-9 The impact of maternal and fetal blood types on umbilical cord nucleated red blood cell counts has not been reported to date. Hemolytic disease of the newborn rarely occurs with maternal-fetal ABO incompatibility.10 ABO incompatibilFrom the Department of Obstetrics and Gynecology, University of Wisconsin Medical School–Milwaukee Clinical Campus. Received for publication July 15, 1999; revised March 8, 2000; accepted April 5, 2000. Reprint requests: Kathleen M. Hanlon-Lundberg, MD, University of Michigan Medical Center, Department of Obstetrics and Gynecology, F4835, Mott Hospital, Box 0264, Ann Arbor, MI 48109. Copyright © 2000 by Mosby, Inc. 0002-9378/2000 $12.00 + 0 6/1/107785 doi:10.1067/mob.2000.107785

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ity usually produces relatively mild hemolysis, if any, because A and B antigen expressions are relatively poorly developed in the fetus and neonate, antigen density is lower than later in life, and the antigens are widely distributed in the tissues. Also, naturally occurring maternal anti-A and anti-B antibodies may be of the immunoglobulin M type, which does not cross the placenta. Hydrops fetalis rarely if ever occurs, and anemia at birth is seldom more than moderate. In this study we sought to evaluate the role of maternal-fetal ABO incompatibility in intrauterine hematopoiesis, as reflected by umbilical cord nucleated red blood cell counts. As a correlate we evaluated some neonatal outcomes associated with maternalfetal ABO incompatibility. Material and methods The Institutional Review Board of Sinai-Samaritan Medical Center, Milwaukee, Wisconsin, approved this study. We prospectively studied pregnant women delivered of live-born infants of ≥37 completed weeks’ gesta-

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tion at our institution between August 1, 1996, and February 28, 1997. After delivery, blood was drawn from the umbilical vein and nucleated red blood cells were manually counted in our hospital laboratory as previously described.11 We obtained maternal data and information concerning neonatal outcomes from medical records, including our institutional perinatal database (Paradox version 5 for Windows; Inprise Corporation, Scotts Valley, Calif). Managing providers determined gestational age to the last completed week of gestation according to clinical and ultrasonographic parameters. Maternal race was according to self-report. ABO testing and antibody screens were performed for all infants delivered during the study period. Direct Coombs testing was performed on all specimens, and specimens with positive results were evaluated for identifiable antibodies. Statistical analysis was done with the SAS statistical software package (version 6.12; SAS Institute, Inc, Cary, NC). The Student t test was used to determine difference of means, and χ2 tests were used for goodness of fit, as appropriate; the Bonferroni correction was used to adjust for multiple comparisons. Significance was defined at P < .05. Parametric results were confirmed by nonparametric analyses (Kruskal-Wallis test), with additional tests of multiple comparisons for nonparametric data as described by Gibbons.12 Results A total of 1948 live-born term neonates were delivered at our institution during the study period. Two hundred eighty-seven cases were excluded from analysis because of technically unacceptable blood specimens or missing data. Complete data on nucleated red blood cell counts and maternal and neonatal blood types were available in 1661 cases, which were considered for further analysis. We compared cases excluded with those included and found no significant differences in maternal age or race, neonatal birth weight, gestational age, or neonatal intensive care unit admission. Demographic characteristics of the study population are reported in Table I. A close correlation between nucleated red blood cells per 100 white blood cells and absolute nucleated red blood cell count was observed (r = 0.904; P < .001). Counts are reported as nucleated red blood cells per 100 white blood cells. We found a mean (±SD) of 9.29 ± 18.56 nucleated red blood cells per 100 white blood cells (range, 0-327 nucleated red blood cells per 100 white blood cells). Data were not normally distributed but rather were skewed toward the lower values. There were 10 outlying values of >100 nucleated red blood cells per 100 white blood cells. All cases, including those with outlying values, were included in the analysis reported here. In our population 20.5% of maternal-fetal dyads (n = 340) had ABO incompatibility (types O and A, n = 136; types O and B, n = 102; types A and B, n = 25; types A and

Hanlon-Lundberg and Kirby 1533

Table I. Demographic characteristics Gestational age (wk, mean ± SD) Birth weight (g, mean ± SD) Maternal age (y, mean ± SD) Maternal race (No.) Black White Hispanic, nonblack Asian Native American Gravidity (No.) 1 2-4 ≥5

39.1 ± 1.2 3283 ± 472 23.6 ± 6.1 1140 (68.6%) 231 (13.9%) 187 (11.3%) 92 (5.5%) 11 (0.7%) 405 (24.4%) 862 (51.9%) 394 (23.7%)

AB, n = 28; types B and A, n = 24; and types B and AB, n = 25). The 590 dyads in which both mother and infant were type O were used as the reference group. Mean nucleated red blood cell counts were lower in ABO-compatible dyads (mean ± SD, 8.29 ± 12.84 nucleated red blood cells per 100 white blood cells; range, 0-216 nucleated red blood cells per 100 white blood cells) than in ABOincompatible dyads (mean ± SD, 13.16 ± 13.16 nucleated red blood cells per 100 white blood cells; range, 0-327 nucleated red blood cells per 100 white blood cells; P = .006). In comparison, neonates born to mothers with A and B antigens had significantly lower nucleated red blood cell counts. Specific blood group combinations are reported in Table II. Nonparametric testing (KruskalWallis test) confirmed these findings. Overall, median nucleated red blood cell counts were lower in ABO-compatible dyads (median, 5 nucleated red blood cells per 100 white blood cells; range, 0-216 nucleated red blood cells per 100 white blood cells) than in ABO-incompatible dyads (median, 6 nucleated red blood cells per 100 white blood cells; range 0-327 nucleated red blood cells per 100 white blood cells; P < .01). When each blood group combination was compared with the dyads in which both mother and infant were type O, with the critical value for significance adjusted by means of the Bonferroni correction for multiple comparisons (α = .05/13 = .0038), only the increase in the type O mother and type B neonate combination remained statistically significant (P < .002). Coombs test results were positive for 77 babies. Of these, 61 had mild reactivity (weak to 1+ positive), 14 had moderate reactivity (2+ positive), and 2 had strong reactivity (≥3+ positive). Nonparametric testing was applied to the data and confirmed findings of preliminary parametric testing at a higher level of significance. Infants with positive Coombs test results had elevated nucleated red blood cell counts relative to infants with negative Coombs test results (positive, n = 77; mean ± SD, 28.13 ± 55.23 nucleated red blood cells per 100 white blood cells; range, 0-327 nucleated red blood cells per 100 white blood cells; negative, n = 1584; mean ± SD, 8.37 ± 14.03 nucleated red blood cells per 100 white blood cells;

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Table II. Nucleated red blood cell count according to maternal and neonatal blood types Source

Blood type

No.

Mean ± SD

Median

Range

Mother Neonate

A A AB B O AB A AB B B A AB B O O A B O

442 264 28 25 125 61 26 8 27 330 24 25 170 111 828 136 102 590

7.80 ± 9.66 7.18 ± 8.46 5.78 ± 5.53 8.92 ± 10.06 9.32 ± 12.23 6.28 ± 6.82 5.42 ± 6.85 7.75 ± 7.03 6.67 ± 6.89 8.42 ± 10.77 5.83 ± 7.23 6.12 ± 4.93 9.77 ± 12.57 7.43 ± 9.03 10.65 ± 24.26 10.80 ± 29.36 22.82 ± 45.81 8.51 ± 15.44

4 4 4 5 5 4 2.5 5 4 5 4.5 5 6 4 5 5 9 4

0-82 0-45 0-21 0-40 0-82 0-33 0-27 0-24 0-33 0-84 0-34 0-20 0-84 0-68 0-327 0-327 0-276 0-216

Mother Neonate Mother Neonate

Mother Neonate

Statistical significance

P = .107 P = .029* P = .845 P = .524 P = .042* P = .781 P = .219 P = .102 P = .046* P = .277 P = .314 P = .380 P < .002† Reference

*With Bonferroni correction, P > .05 or not significant. †Significantly greater nucleated red blood cell counts both with and without Bonferroni correction.

Table III. Cases with >100 nucleated red blood cells per 100 white blood cells Nucleated red blood cells/ Maternal Neonatal Case 100 white blood cells blood type blood type Neonatal disposition

Hospital stay (d) Coombs test result

1 2 3 4

216 153 128 327

O O O O

O O O A

Neonatal nursery Neonatal intensive care unit Neonatal nursery Neonatal intensive care unit

2 21 1 5

5 6 7 8 9

276 241 197 152 151

O O O O O

B B B B B

Neonatal intensive care unit Neonatal nursery Neonatal nursery Neonatal nursery Neonatal intensive care unit

7 2 2 1 11

10

101

O

B

Neonatal nursery

range, 0-276 nucleated red blood cells per 100 white blood cells; P < .0001). Neonates with mildly reactive Coombs test results had a mean (±SD) of 13.44 ± 17.17 nucleated red blood cells per 100 white blood cells (range, 0-86 nucleated red blood cells per 100 white blood cells; P < .0001). Neonates with moderately reactive Coombs test results had a mean ± SD of 94.42 ± 103.35 nucleated red blood cells per 100 white blood cells (range, 10-327 nucleated red blood cells per 100 white blood cells; P < .0001). The 2 neonates with strongly reactive Coombs test results had nucleated red blood cell counts of 6 and 18 nucleated red blood cells per 100 white blood cells; statistical analysis of this small number was not meaningful. All infants but 1 with a positive Coombs test result had maternal-fetal ABO incompatibility (76/77; 98.7%). The single neonate with a strongly reactive Coombs test result (4+) and ABO compatibility (both mother and infant type B) was born to an Rh-sensi-

1

Comment

Negative — Negative Down syndrome Negative — Moderately positive Meconium aspiration syndrome Negative Diabetes mellitus Moderately positive Diabetes mellitus Moderately positive — Moderately positive — Moderately positive Hypoxic-ischemic encephalopathy; death Moderately positive —

tized mother. This neonate had 6 nucleated red blood cells per 100 white blood cells, a birth hematocrit of 54% (normal range, 45%-65%), and a maximum bilirubin level of 5.7 mg/dL (normal). This infant was discharged home on the first day after birth. Antibodies were detected in 4 additional neonates, each of whom demonstrated mildly reactive Coombs test results: 2 had anti-Lea antibodies (3 and 86 nucleated red blood cells per 100 white blood cells), 1 had passive anti-D antibodies (6 nucleated red blood cells per 100 white blood cells), and 1 had anti-Fya antibodies (6 nucleated red blood cells per 100 white blood cells). We evaluated the role of Rh incompatibility in our population. The single case of known Rh sensitization was described previously. Of the 1535 Rh-positive mothers, 1467 were delivered of Rh-positive neonates, with a mean (±SD) of 9.47 ± 18.76 nucleated red blood cells per 100 white blood cells (range, 0-327 nucleated red blood cells

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per 100 white blood cells). This group provided reference values. Again, nonparametric testing was performed and confirmed parametric testing results. The remaining 68 Rh-positive mothers were delivered of Rh-negative neonates (mean ± SD, 7.43 ± 9.38 nucleated red blood cells per 100 white blood cells; range, 0-67 nucleated red blood cells per 100 white blood cells; P > .05). Of the 126 Rh-negative mothers, 89 were delivered of Rh-positive neonates (mean ± SD, 8.54 ± 23.07 nucleated red blood cells per 100 white blood cells; range, 0-216 nucleated red blood cells per 100 white blood cells; P > .05). The remaining 37 Rh-negative mothers were delivered of Rhnegative neonates (mean ± SD, 7.43 ± 9.05 nucleated red blood cells per 100 white blood cells; range, 0-44 nucleated red blood cells per 100 white blood cells; P > .05). In summary, infants born to Rh-negative women (both Rh-positive and Rh-negative neonates) had fewer nucleated red blood cells per 100 white blood cells than did infants born to Rh-positive mothers. Ten neonates had >100 nucleated red blood cells per 100 white blood cells (0.6% of a total of 1661). Table III lists the maternal and fetal ABO status of these cases. ABO incompatibility was present in 7 of 10 cases (70%), which is a greater prevalence than in the remaining population (333/1681; 20.2%; P < .01). Additional clinical information on the neonates with outlying values was previously reported elsewhere.13 Comment Nucleated red blood cells are produced in response to increased erythropoietin, which in turn is stimulated by hypoxia. One reason for hypoxia may be fetal anemia. Fetal hemolytic anemia caused by Rh sensitization is known to stimulate erythropoiesis and to increase release of immature red blood cells into the circulation.14 ABO incompatibility may also cause hemolysis, but this is rarely severe enough to cause hydrops fetalis.10 This study is the first to evaluate the role of maternal-fetal ABO compatibility in determination of umbilical cord nucleated red blood cell counts. Our patient demographic characteristics reflect the diverse population that our central city hospital serves. In a previous study we established normative data for nucleated red blood cell counts in singleton term neonates in our population.13 In that series of 1112 cases we found nucleated red blood cells to be more common in the circulation in term neonates than had previously been reported. This study confirms that finding. As anticipated, ABO incompatibility was strongly associated with an elevation in nucleated red blood cell count. The clinical courses of neonates with elevated nucleated red blood cell counts as a result of ABO incompatibility were mild, as anticipated on the basis of previous neonatal research that has suggested a usually mild course for neonates affected by ABO incompatibility.10 Type B in-

fants born to type O mothers had the highest nucleated red blood cell counts and were overly represented in the group admitted to the neonatal intensive care unit. Conversely, the nucleated red blood cell counts of type A infants born to type O mothers were not significantly elevated relative to dyads in which both mother and infant were type O. This finding is puzzling, because blood group substance A1 has been reported to be the most strongly antigenic and has been associated with the greatest number of cases of clinically apparent hemolysis.10 The 10 neonates with >100 nucleated red blood cells per 100 white blood cells had a higher incidence of ABO incompatibility than did the total study population. Of these, 2 were born after pregnancies complicated by diabetes mellitus, a condition known to be associated with increased nucleated red blood cells. Four neonates were admitted to the neonatal intensive care unit. Neonatal intensive care unit admission was related to the following situations: (1) Down syndrome with hypocontractile cardiac function and respiratory distress, (2) mild meconium aspiration syndrome with ABO incompatibility and a peak bilirubin level of 10.9 mg/dL, (3) respiratory distress in a 4810-g neonate born to a mother with diabetes mellitus at 37 weeks’ gestation, and (4) severe hypoxic-ischemic encephalopathy culminating in death. These 4 neonatal intensive care unit admissions represented the only infants with outlying values who stayed in the hospital for >2 days. The other 6 of the 10 neonates with >100 nucleated red blood cells per 100 white blood cells went home with their mothers 1 to 2 days after birth. In this study we demonstrated that ABO incompatibility was associated with an elevation in nucleated red blood cell count in term neonates. Even with extremely high nucleated red blood cell counts, however, neonatal outcome was generally good unless there were complications by other factors. There was great overlap in nucleated red blood cell counts between neonates affected by ABO incompatibility and those not affected. Elevated nucleated red blood cell counts may not always connote a process of long-term neonatal consequence. Other factors contributing to elevated nucleated red blood cell counts and to longer-term outcomes, which may or may not be associated, remain to be elucidated. REFERENCES

1. Javert CT. The occurrence and significance of nucleated erythrocytes in the fetal vessels of the placenta. Am J Obstet Gynecol 1939;37:184-94. 2. Sinha HB, Mukherjee AK, Bala D. Cord blood haemoglobin (including foetal haemoglobin), and nucleated red cells in normal and toxaemic pregnancies. Indian Pediatr 1972;9:540-3. 3. Shivhare K, Chawla K, Khan MA, Mathur PS. Effect of maternal toxaemia on total haemoglobin, foetal haemoglobin and nucleated red blood cells in cord blood. Indian J Pediatr 1976;43: 349-56. 4. Green DW, Mimouni F. Nucleated erythrocytes in healthy infants and in infants of diabetic mothers. J Pediatr 1990;116: 129-31.

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5. Philip AG, Tito AM. Increased nucleated red blood cell counts in small for gestational age infants with very low birth weight. Am J Dis Child 1989;143:164-9. 6. Soothill PW, Nicolaides KH, Campbell S. Prenatal asphyxia, hyperlacticaemia, hypoglycaemia, and erythroblastosis in growth retarded fetuses. BMJ 1987;294:1051-3. 7. Groenenberg IAL, Baefts W, Hop WC, Wladimiroff JW. Relationship between fetal cardiac and extra-cardiac Doppler flow velocity waveforms and neonatal outcome in intrauterine growth retardation. Early Hum Dev 1991;26:185-92. 8. Mimouni F, Miodovnik M, Siddiqi TA, Butler JB, Holroyde J, Tsang RC. Neonatal polycythemia in infants of insulin-dependent diabetic mothers. Obstet Gynecol 1986;68:370-2. 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.

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10. Shurin SB. Hematologic problems in the fetus and neonate. In: Fanaroff AA, Martin RJ, editors. Neonatal-perinatal medicine: diseases of the fetus and infant. 5th ed. Chicago: Mosby–Year Book; 1992. p. 953-8. 11. Hanlon-Lundberg KM, Kirby RS, Gandhi S, Broekhuizen FF. Nucleated red blood cells in cord blood of singleton term neonates. Am J Obstet Gynecol 1997;176:1149-56. 12. Gibbons JD. Nonparametric methods for quantitative analysis. New York: Holt, Rinehart, and Winston; 1976. p. 181-7. 13. Hanlon-Lundberg KM, Kirby RS. Nucleated red blood cells as a marker of acidemia in term neonates. Am J Obstet Gynecol 1999;181:196-201. 14. Thilaganathan B, Salvesen DR, Abbas A, Ireland RM, Nicolaides KH. Fetal plasma erythropoietin concentration in red blood cell-isoimmunized pregnancies. Am J Obstet Gynecol 1992;167: 1292-7.