Isoimmunization Is Unlikely to Be the Cause of Hemolysis in
ABO-Incompatible but Direct Antiglobulin Test-Negative Neonates
Marguerite Herschel, MD*; Theodore Karrison, PhD‡; Ming Wen, MS‡; Leslie Caldarelli, MD*; and Beverly Baron, MD§
ABSTRACT. Objective. It is stated that the direct an-tiglobulin (Coombs’) test (DAT) may be negative in ABO hemolytic disease of the newborn. Thus, significant jaundice in neonates who are A-B incompatible with their mothers but DAT test negative is often attributed to isoimmunization and another diagnosis is not sought. We wished to determine the rate of bilirubin production, as an objective measure of hemolysis, in 2 groups of DAT-negative neonates—ABO-compatible and ABO-in-compatible—and in DAT-positive ABO-incompatible neonates.
Methods. In consecutive, term, healthy newborns who were admitted to the general care nursery, we mea-sured the level in parts per million (ppm) of end-tidal breath carbon monoxide (CO), corrected for inspired CO (ETCOc), an index of the rate of bilirubin production. We compared the levels in DAT-negative ABO-incompatible neonates with those in ABO-compatible neonates and with the levels in DAT-positive ABO-incompatible neo-nates. Statistical analysis was performed using 2-sample
tand2tests.
Results. There was no significant difference between the mean 12-hour ETCOc levels in DAT-negative ABO-incompatible neonates (n ⴝ 60, 2.2 ⴞ 0.6 ppm) versus DAT-negative ABO-compatible neonates (nⴝ171, 2.1ⴞ 0.6 ppm), although there was a difference between the mean levels in DAT-positive ABO-incompatible neo-nates (n ⴝ 14, 3.4 ⴞ 1.8 ppm) and the DAT-negative groups. Four DAT-negative ABO-incompatible neonates had elevated ETCOc levels; in 2, we diagnosed a specific hematologic abnormality, namely, glucose-6-phosphate dehydrogenase deficiency in 1 and elliptocytosis in the other.
Conclusion. In DAT-negative newborns with signifi-cant jaundice or increased bilirubin production, even if ABO-incompatible, a cause other than isoimmunization should be sought.Pediatrics2002;110:127–130;direct an-tiglobulin test, Coombs’ test, end-tidal carbon monoxide, neonatal jaundice, ABO incompatibility.
ABBREVIATIONS. DAT, direct antiglobulin test; IgG, immuno-globulin G; ETCOc, end-tidal carbon monoxide corrected for am-bient CO; G6PD, glucose-6-phosphate dehydrogenase.
I
t is commonly held that “the Coombs’ test may be negative in ABO hemolytic disease,”1 although much of the evidence for this opinion is based on older literature from the 1950s and methodology that may no longer be used.2 To investigate this issue, Alter et al3attempted to define the clinical value of the DAT in ABO hemolytic disease as performed by blood bank technicians using routine tube method-ology in the late 1960s. He retrospectively studied the records of 1473 group O mothers and their in-fants, analyzing the hemoglobin, reticulocytes, and bilirubin. Serum bilirubin results were not controlled for the hours of age of the neonates; thus, he ac-knowledged difficulty in their interpretation. When the newborns of O-A and O-B pregnancies with neg-ative DATs were compared with newborns of O-O pregnancies, no significant differences in measures of hemolysis were found. However, when these groups were compared with newborns of the same blood groups with positive DATs, significant differ-ences were noted. Newborns of ABO-heterospecific pregnancies with a negative DAT showed no clinical evidence for greater hemolysis than the homospecific group.It has been shown that A and B antigenic sites are weak on the newborn red blood cell membrane.4 Bowman5pointed out that because there is very little anti-A or anti-B antibody on the neonatal red blood cell in ABO hemolytic disease, the cord blood DAT in this condition is only weakly positive and may be negative unless a sensitive test is used. One currently accepted method in hospital laboratories for per-forming the DAT is a gel test6that is more sensitive than formerly used techniques for detecting immu-noglobulin G (IgG) coating of newborn red cells.
Nonetheless, significant jaundice in DAT-negative neonates with ABO incompatibility (mother blood group O, infant A or B) is still often attributed to isoimmunization. Thus, another cause of the jaun-dice is not sought. As unconjugated elevated biliru-bin levels can be a result of either or both increased bilirubin production or reduced elimination, finding significant jaundice does not provide clear evidence of hemolysis. We were interested to know whether there was a higher rate of hemolysis, as determined by end-tidal carbon monoxide corrected for ambient air (ETCOc), a measure of bilirubin production, in a group of DAT-negative ABO-incompatible neonates compared with DAT-negative ABO-compatible neo-nates; if so, then this would provide evidence to
From the Departments of *Pediatrics, ‡Health Studies, and §Pathology, the University of Chicago Pritzker School of Medicine, Chicago, Illinois. Received for publication Oct 5, 2001; accepted Jan 28, 2002.
Reprint requests to (M.H.) MC 1051, Department of Pediatrics, University of Chicago Children’s Hospital, 5841 South Maryland Ave, Chicago, IL 60637. E-mail: mhersche@midway.uchicago.edu
support the common idea that isoimmunization leading to hemolysis may occur with a negative DAT. We confined our study to group O mothers because DAT-positive ABO incompatibility with group A or B mothers is rare.7–9
METHODS
We studied consecutive, term, healthy neonates in our general care nursery after obtaining parental written consent. Cord-blood group, Rh type, and DAT were determined on all neonates, ac-cording to the standard practice in our hospital. The DAT was performed using the ID-Micro Typing System (Micro Typing Sys-tems, Inc, Pompano Beach, FL) with the MTS Anti-IgG Card, a sensitive technique for identifying IgG coated red blood cells. Using the Natus CO-Stat End Tidal Breath Analyzer (Natus Med-ical Inc, San Carlos, CA) for the noninvasive measurement of ETCOc, we determined ETCOc levels at 12⫾6 hours and at 24⫾
6 hours of age. The result is printed out on a data strip and has a reported accuracy of ⫾0.3 parts per million (ppm) (orl/L) or 10%, whichever is greater (product information). The clinical care-givers were not aware of the ETCOc results.
We compared ETCOc levels in DAT-negative group A or B infants born to O mothers with ETCOc levels in DAT-negative group O infants of O mothers. Only the infants of nonsmoking mothers were included in the analysis of the 12-hour ETCOc levels to eliminate confounding by passively acquired CO (in utero) in the fetus. By the 24-hour measurement, the effect of maternal smoking on ETCOc levels is no longer seen.11According to our usual clinical practice, all infants had a bilirubin determination just before hospital discharge or sooner, as clinically indicated. Most determinations were made using the Bilichek device (Respi-ronics, Marietta, GA). Significant jaundice was defined as bilirubin level greater than the 75th percentile on the Bhutani12nomogram of serum bilirubin level for hours of age, a high-risk group for subsequent severe hyperbilirubinemia.13According to our usual practice, infants whose bilirubin level met the American Academy of Pediatrics’ criteria for phototherapy14 had a complete blood count, blood smear for red cell morphology, and a reticulocyte count performed, and, in some cases, a quantitative G6PD enzyme activity level. It was not part of the study design to measure glucose-6-phosphate dehydrogenase (G6PD) levels prospectively in all neonates. A pediatric hematologist reviewed the abnormal blood smears.
Statistical Methods
Comparisons of ETCOc levels among DAT-negative ABO-com-patible, DAT-negative ABO-incomABO-com-patible, and DAT-positive ABO-incompatible groups were performed using 2-samplettests. Comparisons of the proportion of infants with significant jaundice among these same groups were performed using 2tests. The study was approved by the Institutional Review Board of The University of Chicago Hospitals.
RESULTS
During a 15-week period, 660 of 680 consecutive, term newborns who were admitted to the general care nursery of our university inner-city hospital
were enrolled. The population was 83% black; 18% of mothers smoked; 323 (48.9%) mothers had blood group O; 248 DAT-negative neonates were born to nonsmoking, blood group O mothers; 17 of the 248 had missing 12-hour ETCOc values, thus leaving 231 for study at 12 hours. The distribution according to ABO groups is shown in Table 1. The 24-hour values include the DAT-negative infants of all group O mothers (n⫽303); 41 ETCOc values were missing at 24 hours, leaving 262 DAT-negative neonates for study. There was no significant difference between the mean of 12-hour ETCOc levels in DAT-negative ABO-incompatible neonates versus ABO-compatible neonates (observed difference: 0.1 ppm; 95% confi-dence interval: ⫺0.2– 0.2 ppm). The distribution of the values in the 2 groups was similar (see Fig 1). There was also no significant difference at 24 hours. In addition, we compared ETCOc levels among the significantly jaundiced infants between ABO-com-patible and ABO-incompatible DAT-negative groups. We found no significant difference (mean⫾ standard deviation: 2.78⫾0.78 in the ABO-compat-ible group versus 2.81⫾0.53 in the ABO-incompat-ible group;P⫽.91), although the sample sizes were small (n⫽ 17 and 10, respectively).
An additional 19 ABO-incompatible neonates of group O mothers were DAT positive. Their mothers’ indirect antiglobulin tests were negative. In those with ETCOc measurements, born to nonsmoking mothers, there was a significant difference in mean 12-hour ETCOc levels between DAT-positive (n ⫽ 14) and DAT-negative (n ⫽ 60) ABO-incompatible neonates. At 24 hours, the difference in the ETCOc levels for DAT-positive versus DAT-negative neo-nates was nearly significant. The plot of ETCOc val-ues according to the 3 DAT groups at 12 hours is shown in Fig 1.
Significant jaundice was noted in 19 of 185 (10.3%) DAT-negative ABO-compatible neonates and in 11 of 63 (17.5%) DAT-negative ABO-incompatible neo-nates; the difference was not significant (P ⫽ .13). However, of the 16 DAT-positive ABO-incompatible neonates born to nonsmoking mothers, 9 (56.2%) had significant jaundice (P ⬍ .001 as compared with DAT-negative groups).
Thirteen DAT-negative neonates of nonsmoking group O mothers had evidence of increased bilirubin production (12-hour ETCOcⱖ3.1 ppm; 3.2 ppm was the 95th percentile for the whole nonsmoking
popu-TABLE 1. ETCOc in ppm (mean⫾SD)
12-Hour ETCOc* PValue 24-Hour ETCOc† PValue DAT-negative
ABO-incompatible‡ (n⫽60) 2.2⫾0.6 (n⫽70) 1.8⫾0.5
.38 .16
ABO-compatible§ (n⫽171) 2.1⫾0.6 (n⫽192) 1.7⫾0.5 DAT-positive
ABO-incompatible‡ (n⫽14) 3.4⫾1.8 .02㛳 (n⫽12) 2.3⫾0.8 .07㛳 SD indicates standard deviation.
* Neonates of nonsmoking mothers.
† Discrepancies in counts of subjects at 24 hours are attributable to missing ETCOc values. ‡ Mother blood group O, neonate group A or B.
§ Mother blood group O, neonate group O.
㛳Compared with DAT-negative ABO-incompatible neonates.
lation,n⫽500) Of interest, 4 of the 13 were found to have a specific hematologic abnormality (Table 2). Of the remaining 9 neonates without a specific hemato-logic diagnosis, only 2 were ABO-incompatible. Of those 2, 1 had significant jaundice but did not require phototherapy; no diagnostic workup was performed in either case. In the ABO-compatible group, 5 of 7 had significant jaundice; 2 of the 5 received photo-therapy. Their workup was normal, including the G6PD levels. The remaining neonates had no diag-nostic work-up.
DISCUSSION
Our data, which are based on the objective measure-ment of the rate of bilirubin production using ETCOc in unselected neonates admitted to our general care nurs-ery, show that there is no statistically significant in-crease in bilirubin production in DAT-negative
incompatible neonates as compared with ABO-compatible neonates when using the gel technique6for DAT testing. This supports the conclusion of Alter et al3 that there is no clinically detectible difference in measures of hemolysis in DAT-negative newborns from ABO-incompatible compared with ABO-compat-ible pregnancies.
It is well known that there is a wide spectrum in the manifestations of ABO hemolytic disease of the newborn, with the possibility of there being some correlation of the severity of disease with the strength of the DAT. Desjardins et al15 found that with clinically significant ABO hemolytic disease for which the criteria for the diagnosis were strict, the DAT was positive. Thus, they agreed with Alter et al3 that in clinically significant ABO hemolytic disease, one would expect the DAT to be positive.
In the context of our results, the findings of Kaplan
Fig 1. Distribution of ETCOc values according to DAT groups: neonates of nonsmoking mothers.
TABLE 2. Four DAT-Negative Neonates With a Specific Hematologic Diagnosis (Nonsmoking Mothers) Maternal Blood
Group
Neonatal Blood Group
ETCOc ppm (Hours of Age)
Hematocrit (%)
Reticulocyte (%)
Bilirubin mg% (Hours of Age)
Diagnosis
O A 3.4 (8) 36 Not 15.8 (52) G6PD
2.3 (20) done 20.7 (59) deficiency (male)
O A 3.7 (17) 30 11 12.4 (40)
3.4 (25) Elliptocytosis
O O 3.8 (10) 56 5 10.7 (21) G6PD
3.2 (20) deficiency (male)
O O 3.1 (8) 63 8 14.8 (27) Heterozygous G6PD
2.5 (25) deficiency (female) and
et al16 are interesting. They studied the relationship of Gilbert’s syndrome and hyperbilirubinemia (as defined as bilirubin levelⱖ15 mg % in the first week of life) in a population of DAT-negative ABO-incom-patible and ABO-comABO-incom-patible neonates. The incidence of hyperbilirubinemia was significantly higher only in the subgroup of DAT-negative ABO-incompatible neonates who were homozygous for a variant in the uridine diphosphate glucuronosyltransferase gene promoter known as Gilbert’s syndrome; this causes diminished activity of the bilirubin conjugating en-zyme. The incidence of hyperbilirubinemia in DAT-negative ABO-incompatible neonates without Gil-bert’s syndrome was no different from that in ABO-compatible newborns. The method of DAT testing in Kaplan’s study was not given, and their definition of hyperbilirubinemia was different from our designa-tion of significant jaundice.
It seems that the DAT, as determined by the gel test,6 will be positive in neonates who have ABO incompatibility and clinically significant hemolysis as a result of isoimmunization. On the basis of our data, we conclude that in infants who are DAT neg-ative (as determined by a sensitive test) with signif-icant jaundice or increased bilirubin production, a cause other than isoimmunization should be sought, even if there is ABO incompatibility with the moth-er’s blood group, as there is a possibility of finding a specific hematologic disorder, for example, G6PD deficiency or a red blood cell membrane defect. As our population is predominantly black, it would be of interest to know whether our results are applica-ble to other populations.
ACKNOWLEDGMENTS
The study was supported by equipment and a grant from Natus Medical, Inc, and, in part, at the Scripps Research Institute by National Institutes of Health grant HL 25552-10.
We thank Ernest Beutler, MD, of the Scripps Research Institute (La Jolla, CA) for G6PD and UGT1A1 promoter genotype analysis of the female neonate, case 4.
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DOI: 10.1542/peds.110.1.127
2002;110;127
Pediatrics
Baron
Marguerite Herschel, Theodore Karrison, Ming Wen, Leslie Caldarelli and Beverly
but Direct Antiglobulin Test-Negative Neonates
Isoimmunization Is Unlikely to Be the Cause of Hemolysis in ABO-Incompatible
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DOI: 10.1542/peds.110.1.127
2002;110;127
Pediatrics
Baron
Marguerite Herschel, Theodore Karrison, Ming Wen, Leslie Caldarelli and Beverly
but Direct Antiglobulin Test-Negative Neonates
Isoimmunization Is Unlikely to Be the Cause of Hemolysis in ABO-Incompatible
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