Hemolysis and Hyperbilirubinemia in Direct ABO Blood Group Heterospecic Neonates

Michael Kaplan, MB, ChB, Cathy Hammerman, MD, Hendrik J. Vreman, PhD, Ronald J. Wong, BSc, and David K. Stevenson, MD Objective We quantied hemolysis and determined the incidence of hyperbilirubinemia in neonates who were direct antiglobulin titer (DAT)-positive, ABO heterospecic, and compared variables among O-A and O-B subgroups. Study design Plasma total bilirubin (PTB) was determined before the neonates were discharged from the hospital and more frequently when clinically warranted, in neonates who were DAT positive with blood group A or B and with mothers who had blood group O. Heme catabolism (and therefore bilirubin production) was indexed by blood carboxyhemoglobin corrected for inspired carbon monoxide (COHbc). Hyperbilirubinemia was dened as any PTB concentration >95th percentile on the hour-of-life-specic bilirubin nomogram. Results Of 164 neonates, 111 were O-A and 53 O-B. Overall, hyperbilirubinemia developed 85 neonates (51.8%), and it tended to be more prevalent in the O-B neonates than O-A neonates (62.3% versus 46.8%; P = .053). Hyperbilirubinemia developed in more O-B newborns than O-A newborns at <24 hours (93.9% versus 48.1%; P< .0001). COHbc values were globally higher than our previously published newborn values. Babies in whom hyperbilirubinemia developed had higher COHbc values than the already high values of babies who were nonhyperbilirubinemic, and O-B newborns tended to have higher values than their O-A counterparts. Conclusions DAT-positive, ABO heterospecicity is associated with increased hemolysis and a high incidence of neonatal hyperbilirubinemia. O-B heterospecicity tends to confer even higher risk than O-A counterparts. (J Pediatr 2010;-:---).

BO blood group heterospecic (mother group O, newborn group A or B) newborns are at risk for hyperbilirubinemia caused by immune-based hemolysis.1 The hemolysis occurs when maternal immunoglobulin G anti-A or anti-B antibodies cross the placenta and attach to the apposite antigen site on the neonatal red cell. Resultant heme catabolism increases bilirubin production. For each molecule of bilirubin produced, equimolecular quantities of carbon monoxide (CO) are produced. Carboxyhemoglobin (COHb) quantication may index the rate of bilirubin production.2 The direct antiglobulin titer (DAT) test is regarded as the cornerstone of diagnosis of immune hemolytic disease of the newborn.1 Some reports suggest that this test may be only a weak predictor of severe hyperbilirubinemia3,4 and encountered only infrequently in infants re-admitted to the hospital for jaundice.5 However, these reports contrast with the prominence of cases of ABO heterospecicity in recent communications on severe hyperbilirubinemia, bilirubin encephalopathy, or both.6-9 Furthermore, blood group incompatibility with positive DAT results is listed by the Subcommittee on Hyperbilirubinemia of the American Academy of Pediatrics (AAP) as a major risk factor for the development of severe hyperbilirubinemia and also as a risk factor for neurotoxicity.10,11 ABO heterospecicity therefore may have serious consequences. To capture bilirubin dynamics during the rst days of life, the 95th percentile on the hour-of-life-specic nomogram has been used to dene hyperbilirubinemia.12-15 Furthermore, total bilirubin concentrations greater than this percentile may be predictive of severe hyperbilirubinemia. Our objective was to reevaluate the contribution of DAT-positive ABO heterospecicity to neonatal hyperbilirubinemia by assessing the incidence of jaundice with this new denition of hyperbilirubinemia. We further assessed the incidence of hyperbilirubinemia occurring in <24 hours, which may be indicative of hemolysis,10 and compared the risk of hemolysis and hyperbilirubinemia between O-A and O-B subgroups. We quantied the degree of hemolysis by measuring blood COHb corrected for inhaled (ambient) CO (COHbc).2
From the Department of Neonatology, Shaare Zedek Medical Center, Jerusalem, Israel (M.K., C.H.); Faculty of Medicine, The Hebrew University, Jerusalem, Israel (M.K., C.H.); and Department of Pediatrics, Stanford University School of Medicine, Stanford, CA (H.V., R.W., D.S.) Supported at Stanford University by the National Institutes of Health (grants RR00070 and RR025744), the Hess Research Fund, the HM Lui Research Fund, and the Mary L Johnson Research Fund. The authors declare no conicts of interest.
0022-3476/$ - see front matter. Copyright 2010 Mosby Inc. All rights reserved. 10.1016/j.jpeds.2010.05.024

AAP CO COHb COHbc DAT G-6-PD PTB tHb

American Academy of Pediatrics Carbon monoxide Carboxyhemoglobin Carboxyhemoglobin corrected for inspired CO Direct antiglobulin titer Glucose-6-phosphate dehydrogenase Plasma total bilirubin Total hemoglobin

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Methods
The study was approved by the institutional review board of the Shaare Zedek Medical Center. Because of the benign nature of the study, which did not involve randomization or administration of a study drug, oral parental consent only was required. The clinical wing of the study was conducted in the well-infant nurseries of the Shaare Zedek Medical Center from January 2006 to April 2007. A sample of consecutive (except for the conditions mentioned below) infants who were DAT-positive with blood group A or B who were born at $37 weeks gestation to mothers with blood group O was selected for enrollment in the study shortly after birth. To avoid any inclusion bias, patients were included before any plasma total bilirubin (PTB) results were available. Neonates with any obvious condition likely to increase jaundice, other than ABO incompatibility, such severe bruising, sepsis, Down syndrome, glucose-6-phosphate dehydrogenase (G6-PD) deciency, or positive DAT results from any cause other than ABO isoimmunization, were excluded. Similarly, newborns who were DAT-positive and ABO heterospecic and were also Rh-positive and born to Rh-negative mothers were excluded because of the difculty in differentiating a positive DAT caused by Rh isoimmunization from that caused by ABO heterospecicity. Newborns were not enrolled in the study on weekends or secular or religious holidays. Routine treatment of infants born to blood group O mothers at the Shaare Zedek Medical Center has been described.16 Blood type and DAT tests were routinely performed on cord blood of all infants born to blood group O mothers. Results were available within 24 hours of delivery and frequently sooner. Newborns were assessed visually for jaundice at the time of admission to the nursery and subsequently at least once per nursing shift. PTB testing was performed on any newborn with jaundice appearing within the rst 24 hours and after that period, as clinically warranted. During routine, predischarge metabolic screening, a PTB determination was performed on infants who were DAT positive. All PTB results were plotted on the hour-of-life-specic bilirubin nomogram, and the percentile and risk category were determined. Phototherapy for DAT-positive infants was instituted in accordance with the 2004 AAP guidelines for neonates with risk factors.10 After the infants were discharged from the hospital, follow-up PTB determination was performed when necessary on an outpatient basis at our hospital. Indications for follow up were based on the risk category designated with the predischarge PTB concentration, according to the guidelines of the Israel Neonatal Society for the management of neonatal hyperbilirubinemia and prevention of kernicterus.17 Blood sampling for COHb determination was performed before discharge from the hospital at the time of routine metabolic screening. Simultaneous with the COHb sampling, a sample of air from the nursery in which the infant was being cared for was collected for CO analysis. The timing of meta2


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Vol. -, No. bolic screening was suited to COHb sampling of neonates of smoking mothers, because by 48 hours there should no longer be any effect of the smoking on the newborns COHb concentrations.18 COHb Blood for COHb determination (150 mL) was collected in custom-prepared capillary tubes containing heparin and saponin, supplied by Stanford University. The lled tubes were sealed, and the contents were mixed, stored at 18  C, and sent on wet ice to Stanford University. COHb was determined as a percentage of total hemoglobin (tHb) in one batch with a gas chromatographic method.18 The tHb latter was measured by using the same blood sample with a cyanmethemoglobin method.18 The within-day and between-day coefcients of variation for this method for reference blood samples are 3% and 8%, respectively. CO content of the sampled ambient air was measured at Shaare Zedek Medical Center by using a CO analyzer supplied for this purpose by Stanford University, and measured COHb values were corrected for inspired CO to derive COHbc, as described.18 Previously published COHbc and tHb values for neonates from this nursery that were analyzed with the same methodology by the Stanford University laboratory are supplied for comparison. The reference newborns were not DAT positive or G-6-PD decient.19,20 DAT and Blood Type DAT testing was performed routinely in the blood bank of the Shaare Zedek Medical Center on umbilical cord blood with an agglutination technique and reported on a scale of to ++++ (DiaMed-IDMicroTyping System, ID-Card LISS/Coombs; DiaMed AG, Cressier s/Morat, Switzerland). Blood group typing was performed routinely on umbilical cord blood with standard blood bank techniques. Plasma Total Bilirubin Routine PTB testing was measured on heparinized, centrifuged, capillary tube samples by absorbance of bilirubin at 455 nm (NEO BIL Model A2; Digital and Analog Systems, Rome, Italy). Data Analysis Hyperbilirubinemia was dened as any PTB value >95th percentile on the hour-of-life-specic nomogram.12 COHbc, tHb values, and factors relating to hyperbilirubinemia were compared between the O-A and O-B subgroups and, also within the subgroups, between hyperbilirubinemic and non-hyperbilirubinemic neonates. Categorical variables were compared with c2 analysis. Continuous variables with a normal distribution were compared with the Student t test, and in others a Mann-Whitney rank sum test was used. The incidence of hyperbilirubinemia was compared by calculating the relative risk (95% CI), in which case significance was dened as a 95% CI that did not include one. For other comparisons, signicance was dened as a P value <.05.
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ORIGINAL ARTICLES

Table I. Demographic details of the newborns reported for the entire group and O-A and O-B subgroups
Total Number of neonates (n) Birth weight (g, mean SD) Gestational age (weeks) Male (%) Cesarean section (%) Breast feeding (exclusive or partial, %) Maternal smoking (%) Jewish:Arab (n) 164 3401 425 39 1 44 10 88 4 150 (92%):14 (8%) O-A 111 (67.7%) 3434 450 39 1 47 9 89 4 104 (93%):7 (7%) O-B 53 (32.3%) 3342 359 39 1 41 11 85 6 46 (87%):7 (13%) Signicance O-A versus O-B P = .2 P=1 P = .6 P = .9 P = .6 P = .8 P = .7

Results
One hundred sixty-four newborns who were DAT positive with blood group A or B born to blood group O mothers were enrolled between January 2006 and April 2007 (Table I). Overall, a PTB value >95th percentile for hour-of-life at any point 85 developed in newborns (51.8%). Age at rst PTB >95th percentile was 19 11 hours (range, 1-48 hours; one additional newborn was re-admitted at age 80 hours), with corresponding PTB 9.9 2.5 mg/dL (range, 5.1-17.8 mg/ dL). Early hyperbilirubinemia (PTB >95th percentile during the rst 24 hours) was noted in 56 newborns (34.1% of the cohort, 66.7% of those with hyperbilirubinemia), and in 27 of 56 newborns (48.2%), PTB >95th percentile was recorded within the rst 12 hours. Mean (SD) PTB for the rst reading >95th percentile for the <24-hour group was 8.9 1.9 mg/dL, and the mean PTB for the <12-hour subgroup was 8.4 1.9 mg/dL. Of the remaining 29 neonates with hyperbilirubinemia, all except one became hyperbilirubinemic between 25 and 48 hours (31 6 hours). Phototherapy was administered to 80 neonates of the entire group at an average PTB concentration of 10.6 3.0 mg/dL at a mean age of 22 17 hours. Of the 53 neonates with hyperbilirubinemia at <24 hours of age, phototherapy was administered to 51 (mean PTB, 9.2 2.0 mg/dL at 13 8 hours). All neonates responded to phototherapy. None of the neonates required intravenous immune globulin infusion or exchange transfusion. A comparison between the O-A and O-B subgroups is summarized in Table II. Although there was a trend in the O-B subgroup toward higher risk the development of

hyperbilirubinemia in general, hyperbilirubinemia did develop in signicantly more O-B newborns within the rst 24 hours than in their O-A counterparts. The age at which the rst PTB value >95th percentile was noted was earlier in the O-B neonates. Similarly, there was a trend for phototherapy to be commenced earlier in the OB neonates. COHbc studies for the entire cohort, and for the O-A and O-B subgroups, are presented in Table III. Overall, COHbc values were signicantly higher than those of a previously reported newborn cohort.19 Both in the entire cohort and within the O-A and O-B subgroups, neonates in whom hyperbilirubinemia developed had higher COHbc values than the already high values of neonates who did not have any documented PTB value >95th percentile. Furthermore, the percentage of newborns in whom hyperbilirubinemia developed increased in tandem with increasing COHbc percentile values (Figure). There was a trend for the O-B subgroup to have COHbc values that were higher than the already high levels of the O-A subgroup. tHb values were 17.4 2.9 g/dL for the cohort. These were lower than our previously reported reference values (19.0 2.4 g/dL, P < .001).20 Within the ABO heterospecic cohort, tHb values for the hyperbilirubinemic newborns were lower than for those non-hyperbilirubinemic neonates (16.9 2.9 g/dL versus 18.2 2.7 g/dL, P = .007). Even in those ABO heterospecic neonates who did not become hyperbilirubinemic, tHb values were lower than those of the reference group. Although the tHb values were somewhat lower for the O-B neonates, the difference between them and the O-A group was not signicant (17.0 3.1 g/dL versus 17.7 2.8 g/dL, P = .2).

Table II. Comparison of hyperbilirubinemia and need for phototherapy between neonates with O-A and O-B heterospecicity
Category Number of infants Hyperbilirubinemia (n) Age at rst PTB >95th percentile (hours; mean SD) Hyperbilirubinemia within rst 24 hours (n) First PTB >95th percentile (mg/dL) Phototherapy (n) Age at which phototherapy commenced (hours; median [interquartile range]) PTB at commencement of phototherapy (mg/dL)
Hyperbilirubinemia was dened as any PTB value >95th percentile. RR, relative risk.

O-A 111 52 (46.8%) 20.6 11.5 25/52 (48.1%) 10.2 2.6 48 (43.2%) 21.5 (12-32) 11.2 3.2

O-B 53 33 (62.3%) 15.3 9.8* 31/33 (93.9%)* 9.3 1.9 33 (62.3%) 17 (7-27) 9.8 2.4*

Signicance RR, 1.34; 95% CI, 0.99-1.77; P = .053 P = .035 P < .0001 P = .17 P = .14 P = .07 P = .04

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Table III. Blood carboxyhemoglobin values, corrected for inspired CO (COHbc) for the entire ABO heterospecic group and for the O-A and O-B subgroups individually
COHbc (% tHb) Overall, n COHbc (mean SD) Hyperbilirubinemia, n COHbc Non-hyperbilirubinemia, n COHbc Signicance Entire ABO group 163 1.24 0.40* 85 1.42 0.39 78 1.00 0.25 P < .001 O-A 111 1.20 0.38 52 1.40 0.36 59 0.99 0.26 P < .001 O-B 53 1.32 0.44 33 1.45 0.45 20 1.04 0.24 P = .002 Reference group 131 0.77 0.19

For reference, COHbc values for previously reported, healthy, non-hemolyzing neonates are included.19 *P < .0001, entire group vs reference group. P = .07, O-B vs O-A.

Discussion
Of the neonatal population delivered at the Shaare Zedek Medical Center, 21% comprise blood group A or B newborns born to group O mothers, and 15% of them are DAT positive.16 In this study, we documented a 52% incidence of hyperbilirubinemia in the DAT-positive subgroup. This incidence is clearly many-fold that of other population groups studied with the identical denition of hyperbilirubinemia. For example, in a multicenter, multinational study of 1370 newborns, hyperbilirubinemia developed in 8.8%.13 In an African-American male cohort, hyperbilirubinemia was documented in 6.7% of 436 G-6-PD normal, control infants and 21.9% of newborns who were 64 G-6-PD decient.14 Further adding to the high risk nature of these infants is the high incidence of hyperbilirubinemia occurring within the rst 24 hours. However, in almost all babies in whom hyperbilirubinemia developed it did so within the rst 48 hours, implying little risk of subsequent hyperbilirubinemia in those discharged after that time. Many of the babies had combinations of conditions listed by the AAP as major risk factors for the

Figure. Incidence of hyperbilirubinemia, dened as any plasma total bilirubin value >95th percentile on the hour-oflife-specic bilirubin nomogram, graded with corrected carboxyhemoglobin (COHbc) percentile value. COHbc percentile ranges (% tHb): <50th percentile, 0.54 to 1.19; 50th to 74th percentile, 1.20 to 1.44; 75th to 90th percentile, 1.45 to 1.76; >90th percentile, 1.79 to 2.6.
4

development of severe hyperbilirubinemia: predischarge bilirubin value in the high risk zone, hemolysis caused by blood group incompatibility with positive DAT, jaundice appearing within the rst 24 hours, and exclusive breast feeding.10 A correspondingly high number of newborns met the requirement for phototherapy. We cannot foretell the bilirubin dynamics had phototherapy not been instituted at low concentrations of PTB in accordance with AAP guidelines. It is possible that in some neonates the serum bilirubin concentrations would have leveled off and not met the criteria for phototherapy at a later age.3,21 Considering that kernicterus is rarely encountered and exchange transfusion currently is unusual, it is not surprising that, despite the high incidence of hyperbilirubinemia, we encountered no cases of this nature. Although our ndings are consistent with earlier reports demonstrating that few DATpositive, ABO-heterospecic neonates will meet the criteria for exchange transfusion,3,5 newborns of these blood group combinations do appear prominently in recent series of severe hyperbilirubinemia and kernicterus.6-9 The apparent mild nature of the disease should not cause complacency for ABO hemolytic disease. There may be an increased effect of hemolytic conditions on the development of bilirubin-induced neurologic dysfunction.22,23 Our elevated COHbc results, comptatible with previously reported data,13,24-26 conrm the hemolytic nature of DAT positive ABO heterospecicity in general and emphasize the high risk nature of these neonates. Despite the overall high rate of hemolysis, not all infants in our series in whom hyperbilrubinemia developed had high levels of COHbc. The percentage of hyperbilirubinemic neonates increased with increasing COHbc percentile values. We speculate that, in the lower percentile groups, varying degrees of immaturity of the bilirubin conjugating system, or presence of the (TA)7 promoter variant of the UGT1A1 gene associated with Gilbert syndrome, with moderately increased heme catabolism, may have contributed to the hyperbilirubinemia.27 However, in those neonates with the highest COHbc levels, hyperbilirubinemia developed in all. In this latter group, the degree of hemolysis must have been sufciently high to overwhelm even the most efcient conjugation process. The literature is inconsistent on the degree of hemolysis and the incidence and severity of hyperbilirubinemia
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- 2010 between O-A and O-B subgroups. Several investigators were unable to show any difference in clinical severity between OA and O-B hemolytic disease of the newborn, although in one report28 there was a trend toward performing exchange transfusion during the rst 24 hours more frequently in OB infants than in O-A infants.29-30 Similarly, a retrospective analysis of ABO hemolytic disease did not nd signicant relationships between the infants blood type and clinical outcome.31 Sisson32 and Kaplan33 reported no signicant differences in severity or response to therapy in the 2 blood types. An infant whose blood group was A was as likely to be affected by ABO hemolytic disease as an infant with blood group B.34 However, Bakkeheim et al found a signicantly increased rate of invasive treatments, including intravenous immune globulin therapy and exchange transfusion, in O-B infants compared with O-A infants.35 Two studies documented a higher need for exchange transfusion in O-B neonates than in O-A neonates.36,37 The reasons for the apparently increased severity of jaundice in the O-B subgroup are not clear. Maternal factors including differing levels of immunoglobulin G anti-A or anti-B may affect the degree of hemolysis, and variations in immunoglobulin G subgroup distribution may alter macrophage-induced red blood cell clearance. The number of fully developed A or B antigen sites on fetal red blood cells may be different, as may be the dilutional effect of other tissues bearing these surface antigens.38 Comparisons of these studies are difcult because inclusion criteria, denitions, therapeutic indications, and time epochs differed from study to study. In contrast, we categorized our patients using an up-to-date denition of hyperbilirubinemia that takes into account the bilirubin dynamics of the rst days of life.12 Standardized criteria for treatment were used,10 and the rate of heme catabolism was assessed with a state of the art method.2 Selection bias was avoided by including all newborns who were DAT positive and ABO heterospecic at birth, before they became jaundiced. Although not all our comparisons achieved statistical significance, O-B heterospecic neonates did appear to be at higher risk than the O-A subgroup. Hyperbilirubinemia did occur earlier in the O-B infants, and hyperbilirubinemia developed in more O-B newborns within the rst 24 hours. This difference was attributable to moderately, although not statistically signicantly, increased hemolysis. Lack of statistical signicance, however, does not preclude an effect of moderately increased hemolysis resulting in increased bilirubin production. Coupled with immaturity of the bilirubin conjugating system, moderately increased hemolysis may have resulted in a higher incidence of hyperbilirubinemia. Shortcomings of our study were the dependence on visual recognition of early jaundice and lack of a xed protocol for PTB determinations during the rst 24 hours. In summary, our study provides information about the incidence, severity, and mechanism of jaundice in ABO heterospecic neonates. A high rate of hemolysis is a hallmark of DAT positivity, with a resultant high incidence of hyperbilirubinemia, especially early hyperbilirubinemia. These factors

ORIGINAL ARTICLES
have the potential of increasing the risk of severe hyperbilirubinemia and bilirubin neurotoxicity. Furthermore, O-B heterospecic neonates appear to be at higher risk than their O-A counterparts n
Submitted for publication Jan 23, 2010; last revision received Apr 8, 2010; accepted May 14, 2010. Reprint requests: Dr Michael Kaplan, Department of Neonatology, Shaare Zedek Medical Center, PO Box 3235, Jerusalem 91031, Israel. E-mail: kaplan@cc.huji.ac.il.

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