Isoimmunization Is Unlikely to Be the Cause of Hemolysis in ABO-Incompatible but Direct Antiglobulin Test-Negative Neonates
* Departments of Pediatrics
Health Studies
Pathology, the University of Chicago Pritzker School of Medicine, Chicago, Illinois
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Objective. It is stated that the direct antiglobulin (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-incompatible—and in DAT-positive ABO-incompatible neonates.
Methods. In consecutive, term, healthy newborns who were admitted to the general care nursery, we measured 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 neonates. Statistical analysis was performed using 2-sample t and 
2 tests.
Results. There was no significant difference between the mean 12-hour ETCOc levels in DAT-negative ABO-incompatible neonates (n = 60, 2.2 ± 0.6 ppm) versus DAT-negative ABO-compatible neonates (n = 171, 2.1 ± 0.6 ppm), although there was a difference between the mean levels in DAT-positive ABO-incompatible neonates (n = 14, 3.4 ± 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 significant jaundice or increased bilirubin production, even if ABO-incompatible, a cause other than isoimmunization should be sought.
Key Words: direct antiglobulin test • Coombs’ test • end-tidal carbon monoxide • neonatal jaundice • ABO incompatibility
Abbreviations: DAT, direct antiglobulin test • IgG, immunoglobulin G • ETCOc, end-tidal carbon monoxide corrected for ambient CO • G6PD, glucose-6-phosphate dehydrogenase
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
It 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 al3 attempted to define the clinical value of the DAT in ABO hemolytic disease as performed by blood bank technicians using routine tube methodology in the late 1960s. He retrospectively studied the records of 1473 group O mothers and their infants, analyzing the hemoglobin, reticulocytes, and bilirubin. Serum bilirubin results were not controlled for the hours of age of the neonates; thus, he acknowledged difficulty in their interpretation. When the newborns of O-A and O-B pregnancies with negative 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 differences 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 Bowman5 pointed 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 performing the DAT is a gel test6 that is more sensitive than formerly used techniques for detecting immunoglobulin 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 jaundice is not sought. As unconjugated elevated bilirubin 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 neonates; if so, then this would provide evidence to 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 |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
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, according to the standard practice in our hospital. The DAT was performed using the ID-Micro Typing System (Micro Typing Systems, 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 Medical 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) (or µl/L) or 10%, whichever is greater (product information). The clinical caregivers 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.11 According 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 (Respironics, Marietta, GA). Significant jaundice was defined as bilirubin level greater than the 75th percentile on the Bhutani12 nomogram of serum bilirubin level for hours of age, a high-risk group for subsequent severe hyperbilirubinemia.13 According 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-compatible, DAT-negative ABO-incompatible, and DAT-positive ABO-incompatible groups were performed using 2-sample t tests. Comparisons of the proportion of infants with significant jaundice among these same groups were performed using 2 tests. The study was approved by the Institutional Review Board of The University of Chicago Hospitals.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
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% confidence 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-compatible and ABO-incompatible DAT-negative groups. We found no significant difference (mean ± standard deviation: 2.78 ± 0.78 in the ABO-compatible group versus 2.81 ± 0.53 in the ABO-incompatible 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 neonates was nearly significant. The plot of ETCOc values 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 neonates; 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 ETCOc 
3.1 ppm; 3.2 ppm was the 95th percentile for the whole nonsmoking population, 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 hematologic 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 phototherapy. Their workup was normal, including the G6PD levels. The remaining neonates had no diagnostic work-up.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Our data, which are based on the objective measurement of the rate of bilirubin production using ETCOc in unselected neonates admitted to our general care nursery, show that there is no statistically significant increase in bilirubin production in DAT-negative ABO-incompatible neonates as compared with ABO-compatible neonates when using the gel technique6 for 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-compatible 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 et al16 are interesting. They studied the relationship of Gilbert’s syndrome and hyperbilirubinemia (as defined as bilirubin level 15 mg % in the first week of life) in a population of DAT-negative ABO-incompatible and ABO-compatible 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 enzyme. The incidence of hyperbilirubinemia in DAT-negative ABO-incompatible neonates without Gilbert’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 designation 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 negative (as determined by a sensitive test) with significant jaundice or increased bilirubin production, a cause other than isoimmunization should be sought, even if there is ABO incompatibility with the mother’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 applicable 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.
|
FOOTNOTES |
|---|
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{at}midway.uchicago.edu
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
- Queenan JT. Erythroblastosis fetalis. In: Fanaroff AA, Martin RJ, eds. Neonatal-Perinatal Medicine: Diseases of the Fetus and Infant. 5th ed. St Louis, MO: Mosby-Year Book, Inc; 1992:242
- Dacie J. Haemolytic disease of the newborn. In: The Haemolytic Anaemias. 3rd ed. London, United Kingdom: Churchill Livingstone; 1999:331–375
- Alter AA, Feldman F, Twersky J, et al. Direct antiglobulin test in ABO hemolytic disease of the newborn. Obstet Gynecol.1969; 33 :846 –851[Web of Science][Medline]
- Romans DG, Tilley CA, Dorrington KJ. Monogamous bivalency of IgG antibodies. J Immunol.1980; 124 :2807 –2811[Abstract]
- Bowman JM. ABO hemolytic disease. In: Creasy RK, Resnik R, eds. Maternal-Fetal Medicine. 4th ed. Philadelphia, PA: WB Saunders; 1999:736–767
- Lapierre Y, Rigal D, Adam J, et al. The gel test: a new way to detect red cell antigen-antibody reactions. Transfusion.1990; 30 :109 –113[CrossRef][Web of Science][Medline]
- Rosenfield RE. A-B hemolytic disease of the newborn.
Blood.1955; 10
:17
–28
[Abstract/Free Full Text] - Ozolek JA, Watchko JF, Mimouni F. Prevalence and lack of clinical significance of blood group incompatibility in mothers with blood type A or B. J Pediatr.1994; 125 :87 –91[CrossRef][Web of Science][Medline]
- Jeon H, Calhoun B, Pothiawala M, Herschel M, Baron BW. Significant ABO hemolytic disease of the newborn in a mother with A2 blood type. Immunohematology.2000; 16 :105 –108
- Vreman HJ, Mahoney JJ, Stevenson DK. Carbon monoxide and carboxyhemoglobin. Adv Pediatr.1995; 42 :303 –334[Medline]
- Herschel M, Karrison T, Wen M, Caldarelli L, Baron B. Evaluation of the direct antiglobulin (Coombs’) test for identifying newborns at-risk for hemolysis as determined by end-tidal carbon monoxide concentration (ETCOc) and comparison of the Coombs’ test with ETCOc for detecting significant jaundice. J Perinatol.2002; 22
- Bhutani VK, Johnson L, Sivieri EM. Predictive ability of a predischarge hour-specific serum bilirubin for subsequent significant hyperbilirubinemia in healthy term and near-term newborns.
Pediatrics.1999; 103
:6
–14
[Abstract/Free Full Text] - Bhutani VK, Gourley GR, Adler S, Kreamer B, Dalin C, Johnson LH. Noninvasive measurement of total serum bilirubin in a multiracial predischarge newborn population to assess the risk of severe hyperbilirubinemia. Pediatrics.2000; 106(2) . Available at: http://www.pediatrics.org/cgi/content/full/106/2/e17
- American Academy of Pediatrics, Committee on Fetus and Newborn. Practice parameter: management of hyperbilirubinemia in the healthy term newborn.
Pediatrics.1994; 94
:558
–565
[Abstract/Free Full Text] - Desjardins L, Blajchman MA, Chintu C, Gent M, Zipursky A. The spectrum of ABO hemolytic disease of the newborn infant. J Pediatr.1979; 95 :447 –449[CrossRef][Web of Science][Medline]
- Kaplan M, Hammerman C, Renbaum P, Klein G, Levy-Lahad E. Gilbert’s syndrome and hyperbilirubinemia in ABO-incompatible neonates. Lancet.2000; 356 :652 –653[CrossRef][Web of Science][Medline]
PEDIATRICS (ISSN 1098-4275). ©2002 by the American Academy of Pediatrics
CiteULike 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
