ABO Hemolytic Disease of the Newborn
A Retrospective Analysis of 254 Cases
D. ROBERT DUFOUR, M.D. AND W. PATRICK MONOGHAN, PH.D.
Dufour, D. Robert and Monaghan, W. Patrick: ABO hemolytic
disease of the newborn. A retrospective analysis of 254 cases.
Am J Clin Pathol 73: 369-373, 1980. ABO hemolytic disease
of the newborn is the single most common cause of neonatal
jaundice, with an incidence of 54.4 per 1,000 births; it occurs
almost exclusively in infants of groups A or B having mothers
of group O. Previous studies have shown a poor correlation
between serologic tests on cord blood and clinical course in
affected infants. In a retrospective analysis of 254 cases of ABO
hemolytic disease of the newborn the relation of laboratory
parameters to incidence and severity of jaundice was studied.
Sixty-five per cent of the infants who had positive direct
antiglobulin tests experienced jaundice, compared with ap-
proximately 35% of control infants or infants who had ABO
hemolytic disease of the newborn with negative direct anti-
globulin test results. Infants who had ABO hemolytic disease
of the newborn with positive direct antiglobulin test results
also had greater severity of jaundice than control infants or
infants who had ABO hemolytic disease of the newborn with
negative direct antiglobulin test results (P < .0001). Thus,
the direct antiglobulin test is a good screening test for ABO
hemolytic disease of the newborn. Sex, race, gravidity, birth
weight, and blood type of the infant did not have significant
relationships to clinical outcome. The combined results of
the direct antiglobulin test and the strength of reaction in a
heat eluate may be of use in prognosis; although differences
were not significant, infants who had stronger eluates had
higher bilirubin values and were more likely to require
therapy. Serologic analysis of cord blood can be useful in the
early detection of infants having the risk of severe jaundice.
(Key words: Erythroblastosis fetalis; Blood group in-
compatibility.)
THE FIRST REPORT of a possible association be-
tween maternal-fetal ABO incompatibility and neonatal
jaundice was made by Halbrecht in 1944.7 Rosenfield12
reported the first controlled study in 1955, and showed
that a positive direct antiglobulin test identified a group
of ABO-incompatible infants showing laboratory evi-
dence of hemolytic disease due to alloantibodies de-
tected on their erythrocytes. Since then, numerous
studies have attempted to establish other laboratory
tests useful in evaluating ABO hemolytic disease; these
Received November 24, 1978; received revised manuscript and
accepted for publication April 9, 1979.
Address reprint requests to Dr. Dufour: LT, MC, USNR,
National Naval Medical Center, Box 399, Bethesda, Maryland
20014.
0002-9173/80/0300/0369 $00.75 American Society of Clinical Pathologists
369
Department of Laboratory Medicine, National Naval
Medical Center, Bethesda, Maryland
studies have recently been reviewed by Gold and
Butler. 5
While it has been recognized that maternal-fetal ABO
incompatibility is the most frequent cause of hemolytic
disease of the newborn, the clinical course is often
benign, and therapy is required in approximately 10%
of the affected infants. 4514 With the declining in-
cidence of Rh hemolytic disease since the introduction
of Rh immune globulin, ABO incompatibility has be-
come the most common cause of hemolytic disease of
the newborn requiring therapy at many institutions.
These observations emphasize the major unsolved
problem in ABO hemolytic disease of the newborn:
while serologic tests (usually the direct antiglobulin
test can detect antigen-antibody interactions, they
may not predict the clinical course of affected infants.
Although the studies of Rosenfield have shown that
infants having a positive direct antiglobulin test result,
as a group, have evidence of increased hemolysis, few
studies have attempted to compare the course of these
infants with that of otherwise normal infants. In an
attempt to evaluate these unanswered questions, a
retrospective study was undertaken to identify any
correlation between clinical severity of ABO hemolytic
disease of the newborn and the various laboratory
parameters tested. This report is an analysis of 254
cases of ABO hemolytic disease of the newborn seen
over a 64-month period.
Materials and Methods
The National Naval Medical Center serves as both
a primary-care hospital for routine obstetric cases and
a tertiary-care center for complicated obstetric cases.
Prenatal blood analysis for each fetus includes ABO-
group and Rh-type determination, and an indirect anti-
human globulin test for detection of atypical allo-
antibodies. A specimen of cord blood, collected by
syringe, is obtained after delivery for each infant and
370 DUFOUR AND MONAGHAN
Table 1. Incidence of Hemolytic Disease
of the Newborn
Total deliveries
Maternal-fetal ABO incompatibility
Mothers of group O
ABO Hemolytic Disease of the Newborn present
ABO Hemolytic Disease of the Newborn absent
Mothers of groups A and B
ABO Hemolytic Disease of the Newborn present
ABO Hemolytic Disease of the Newborn absent
Hemolytic disease other than ABO Hemolytic Disease of the
Newborn
Due to anti-D
Due to other Rh antibodies
Due to non-Rh antibodies (anti-Kell, anti-Jk" twins)
is sent to the Blood Bank for ABO-group, Rh-type,
and direct antiglobulin test determinations. To deter-
mine antibody specificity, a heat eluate is performed
on each cord specimen having a positive direct anti-
globulin test result. All test procedures are listed in
the Technical Manual of the American Association of
Blood Banks. 1
Permanant records of cord-blood results are main-
tained in the Blood Bank; a review of these records
for the previous 64 months indicated a total of 254
cases of ABO hemolytic disease of the newborn. In
addition, the distribution of maternal blood group and
maternal-fetal blood group pairs was tabulated. Criteria
for making this diagnosis varied slightly during the
study. From 1973 to 1976, only infants who had a posi-
tive direct antiglobulin test result and a heat eluate
positive for either anti-A or anti-B or both, were
included. False-negative results had been reported in
ABO hemolytic disease of the newborn; therefore, in
the final 16 months of the study, infants with a heat
eluate positive for either anti-A or anti-B, or both,
were included, regardless of the results of the direct
antiglobulin test.
Hospital records of infants diagnosed as having ABO
hemolytic disease of the newborn were reviewed, and
the following information was obtained: sex, race,
gestational length, gravidity, birth weight, maternal
blood group and type, peak bilirubin level, day of
peak bilirubin, hemoglobin, reticulocyte count, pre-
natal or postnatal complications, and length of hospital
stay. Therapy for hyperbilirubinemia, when given, was
also noted. Immunohematologic analyses recorded for
infants included: blood group and type, direct anti-
globulin test result, and antibody present in the heat
eluate. In the first 24 months of the study, the strength
of reaction in a heat eluate was not graded; in the
last 40 months, it was graded as recommended in the
Technical Manual of the American Association of
Blood Banks. 1 As a control group, all term infants
A.J.C.P. March 1980
admitted during a two-month period were studied in
the same way that patients who had ABO hemolytic
disease of the newborn were studied.
4,706
928 Results
665
252 During the 64 months of the study, there were
413
263 4,949 deliveries. Complete records of maternal and
2 fetal blood groups and types infant direct antiglobulin
261 test results and maternal antibody screens were avail-
able in 4,706 cases (95.1%). The remaining cases most
13 frequently lacked test results of maternal group and
6
4 type; this may have been owing to the transfer of a
3 small percentage of patients from other medical
facilities. The incidence of laboratory evidence of ABO
hemolytic disease of the newborn during the study is
given in Table 1.
Maternal-fetal ABO incompatibility is defined as the
presence of a maternal alloantibody that in reaction
with fetal erythrocytes, could cause agglutination or
hemolysis. Maternal-fetal ABO-incompatible pairs in-
cluded: mothers of group O with infants of group A
or B, mothers of group A with infants of group B or AB
and mothers of group B with infants of group A or AB.
All other maternal-fetal blood group pairs were con-
sidered ABO compatible. Of ABO-incompatible infants
with mothers of group O, 37.9% had laboratory evi-
dence of ABO hemolytic disease of the newborn. Of
ABO-incompatible infants with mothers of groups A or
B, only 0.8% had laboratory evidence of ABO hemo-
lytic disease of the newborn. This difference appears to
be highly significant (x2 = 120.6, P < .000001).
Three sets of infants were evaluated in this study.
The first two sets were of infants showing laboratory
evidence of ABO hemolytic disease of the newborn.
One set consisted of 208 infants who had positive direct
antiglobulin test results and anti-A or anti-B identified
in a heat eluate of cord erythrocytes; the second set
consisted of 38 infants who had negative direct anti-
globulin test results, but who had anti-A or anti-B, or
both, in a heat eluate. The third set (controls) con-
sisted of all 137 full-term infants born in this institution
during a two-month period near the end of the study.
Table 2. Results of Direct Antiglobulin Test in ABO
Hemolytic Disease of the Newborn Correlated
with Clinical Course
Group I
(No Clinical Group 11 Group III
Total Evidence of ("Physiologic (Marked
No. Jaundice) Jaundice") Jaundice)
Positive 208 74 80 54
Negative 38 30 5 3
Control 137 86 42 9
Vol. 73 No. 3 ABO HEMOLYTIC DISEASE OF THE NEWBORN 371
Table 3. Infants Receiving Therapy for ABO Hemolytic Disease of the Newborn
Group Begun
Gravidity and Type Eluate* Therapy on Day
Patient 1, F 3 A Positive Positive Exchange transfusion 2
Patient 2, F 1 A Positive Positive Exchange transfusion 2
Patient 3, F 3 A Positive Positive Phototherapy 4
Patient 4, M 2 A Positive Positive Phototherapy 2
Patient 5, M 1 A Positive 2+ Phototherapy 4
Patient 6, F 1 A Positive 1+ Exchange transfusion, phototherapy 3
Patient 7, F 2 A Positive Positive Phototherapy 1
Patient 8, F 1 A Positive Positive Exchange transfusion, phototherapy 2
Patient 9, F 4 B Positive 2+ Exchange transfusion 3
Patient 10, F 3 B Positive 2+ Exchange transfusion, phototherapy 2
Patient 11, F 3 A Positive 2+ Phototherapy 2
Patient 12, F 2 A Positive 3+ Exchange transfusion 1
Patient 13, M 3 A Positive 2+ Phototherapy, exchange transfusion 3
Patient 14, F 4 A Positive 2+ Exchange transfusion, phototherapy 2
Patient 15, M 2 A Positive 1+ Phototherapy 2
Patient 16, F 3 A Positive Weak positive Phototherapy 3
Patient 17, F 2 B Positive Positive Phototherapy 3
Patient 18, F 1 A Positive Positive Exchange transfusion, phototherapy 1
Patient 19, M 2 B Positive Positive Phototherapy 4
Patient 20, M 3 A Positive 2+ Phototherapy 2
Patient 21, M 3 A Positive 1+ Phototherapy 4
Patient 22, M 1 A Positive 1+ Phototherapy 4
Patient 23, F 2 A Positive Weak Positive Phototherapy 2
Patient 24, F 2 A Negative Weak Positive Phototherapy 3
Patient 25, M 1 A Positive 2+ Phototherapy 1
Patient 26, M 3 B Positive 1+ Exchange transfusion, phototherapy 2
Patient 27, M 2 A Positive 2+ Phototherapy 3
Patient 28, M 2 B Positive Weak positive Phototherapy 2
* Weak positive indicates microscopically positive; positive indicates positive but strength of reaction not given.
The records of all infants were evaluated in the same and marked jaundice (groups II and III) than did either
manner. No significant differences (P > .05) were control infants or infants who had negative direct
found among the three sets of infants when com- antiglobulin test results and positive eluates. Among
parisons were made for sex, race, birth weight, or infants who had positive direct antiglobulin test results,
gravidity of the mother. An additional eight infants 64% had some degree of jaundice and 26% had marked
idenified as having laboratory evidence of ABO hemo- jaundice; this was significantly higher {P < .0001) than
lytic disease of the newborn were not included in this for control infants or infants who had negative direct
study; four infants' charts were not available for re- antiglobulin test results. The incidence of jaundice in
view, and another four infants had coexisting disease infants who had negative direct antiglobulin test results
processes which made evaluation of the ABO hemo- (21% jaundiced, 8% marked) was not significantly
lytic extremely difficult (two being extremely premature, different from that in control infants (37% jaundiced,
one having hereditary spherocytosis, one having Rh 7% marked).
hemolytic disease ofthe newborn). A total of 28 infants (11.4%) required therapy for
Since one of the major manifestations of ABO ABO hemolytic disease. The laboratory and clinical
hemolytic disease of the newborn is hyperbiliru- features of these infants are given in Table 3. Four
binemia, the infants were evaluated for degree of infants received only exchange transfusions: 17 re-
jaundice. Because ofthe wide range of bilirubin values, ceived only phototherapy, and seven received both
it was necessary to group the infants for purposes of exchange transfusions and phototherapy. Although
analysis. Infants who had no clinical evidence of criteria for treatment varied slightly during the course
jaundice were placed in group I; infants who had ofthe study, infants received treatment when the serum
"physiologic" jaundice (bilirubin values less than 12.0 bilirubin level exceeded the albumin binding capacity.
mg/dl), in group II; and infants with jaundice above Most ofthe infants who received exchange transfusions
the physiologic range (marked jaundice), in group III. were treated during the early years of the period
A comparison ofthe courses ofthe three sets of infants studied, before phototherapy was adopted as the
is given in Table 2. As can be seen from the table, preferred primary means of therapy at this institution;
infants who had positive direct antiglobulin test results in the last three years, only infants failing to respond
had a much higher incidence of both "physiologic" adequately to phototherapy, or those having critically
372 DUFOUR AND MONAGHAN
Table 4. Results of Cord Blood Eluates in ABO Hemo-
lytic Disease of the Newborn and Correlation of
Eluate Strength* with Clinical Course
Group I
(No
Clinical Re-
Evidence Group II Group III ceived
of ("Physiologic (Marked Ther-
Jaundice) Jaundice") Jaundice) apy
Weakly positive
(microscopic
orl+) 25 44 25 9 hemolytic disease of the newborn requiring therapy.
Strongly positive
(2-3+) 8 17 14 10
* Etuate strength of reaction was measured by the procedure recommended in the
Technical Methods Manual of the American Association of Blood Banks.
low hemoglobin levels, were treated by exchange
transfusion. In comparison with the remaining infants
who had ABO hemolytic disease of the newborn and
the control group, infants requiring therapy showed no
significant differences of clinical features, with the
exception of the earlier onset of jaundice (x2 = 9.04,
P < .02) and, by definition, more severe jaundice.
Among infants who had positive direct antiglobulin
test results, the strength of the reaction of a heat eluate
was recorded in 133 cases. The clinical course of these
infants is given in Table 4. Infants who had strongly
positive eluates had a slightly higher incidence of
jaundice than those who had weakly positive eluates
and higher mean bilirubin levels (11.3 mg/dl vs. 10.4
mg/dl); however, these differences are not significant.
In addition, a higher proportion of infants who had
strongly positive eluates required therapy than did
infants who had weakly positive eluates (26% vs. 10%,
0.1 > P > .05). Thus the eluate strength appeared to
give additional information to that provided by a
positive direct antiglobulin test result. For infants who
had negative direct antiglobulin test results, the eluate
strength had no relation to the clinical course. Race,
sex, gestational age, birth weight, gravidity of mother,
and blood group also had no relations to the clinical
course. Cord-blood bilirubin, hemoglobin, and reticu-
locyte count were recorded in too few cases to allow
any correlations to be made.
Discussion
ABO hemolytic disease of the newborn occurs
relatively frequently and can be a significant cause of
neonatal morbidity. While the incidence of clinically
significant jaundice in ABO-compatible infants varies
from 6.5% (our series) to 8.4% (other series 9 ), it is
in the range of 25-30% in ABO-incompatible infants
having positive direct antiglobulin test results. 9 In our
A.J.C.P. March 1980
series, ABO hemolytic disease of the newborn was
responsible for jaundice in approximately 20% of all
infants who had clinically significant jaundice; in other
series, it has been as high as 30%. 9 Although ABO
hemolytic disease of the newborn is known to be a
cause of neonatal jaundice, its prevalence as a major
etiologic factor has not previously been emphasized.
Because of the decline in hemolytic disease of the
newborn due to anti-Rh 0 (D), ABO hemolytic disease of
the newborn has become the most common cause of
During the period studied, 28 infants who had ABO
hemolytic disease of the newborn required therapy,
compared with only 13 infants who had hemolytic
disease of the newborn due to any other antibody. This
trend is likely to continue in the future.
ABO hemolytic disease of the newborn is found al-
most exclusively among infants having mothers of
group O. In our series, the incidence of ABO hemolytic
disease of the newborn among ABO-incompatible in-
fants of mothers of group O was 37.9%; this is similar
to the incidence published in previous reports. 411,12
In ABO-incompatible infants of mothers of group A or
B, the incidence was only 0.8%. Rosenfield, in 1955,
was the first to note the marked preponderance of
group O mothers of infants having ABO hemolytic
disease of the newborn; 12 since then, many studies
have been done to try to explain this fact. Rosenfield
and Ohno 13 have shown that, whereas 90% of group O
individuals have IgG anti-A and anti-B, only 34% of
group A or B individuals have IgG anti-B or anti-A. In a
later paper, Kochwa and co-workers 10 showed that,
whereas 70% of group O mothers had titers of IgG anti-
A or anti-B greater than 1:2, no mother of group A or B
did. While this would appear to explain the marked
preponderance of group O mothers of infants having
ABO hemolytic disease of the newborn, it does little to
explain the manner of sensitization. Pregnancy does
not seem to be a major stimulus, since infants of
primigravida mothers may be affected as severely or
more severely than infants of multigravida mothers. 5
Our series confirmed these findings; infants of
multigravida and primigravida mothers were affected
to a similar extent. Of the three women who had more
than one infant during the study, two had infants who
had similar degrees of jaundice: the third mother's first
infant was more severely affected than her second.
There has been much interest in the early prediction
of the occurrence and severity of ABO hemolytic
disease of the newborn. Voak and Bowley14 screened
a large number of women of group O for the presence
of high titer IgG anti-A and anti-B and followed them
to delivery. Although they considered this charac-
teristic to define a "high risk" group, the incidence
Vol. 73 No. 3 ABO HEMOLYTIC DISEASE OF THE NEWBORN
and severity of jaundice in the infants in their study
were not different from those reported by other authors
who studied an unscreened population. Carpella-de
Luca, Pacioni, and Tonelli 3 found a high titer of
IgG anti-A and anti-B to be correlated with a positive
direct antiglobulin test result; however, this high titer
had no apparent correlation with a need for therapy.
Graham, Morrison, and McAndrew 6 used Sephadex
separation of IgG anti-A and anti-B followed by titra-
tion, to define a "high risk" group. However, the
incidence and severity of jaundice in infants in their
screened series were only slightly higher than in infants
in our series, and the differences were not significant.
Thus, at best, prenatal studies have a limited value
in the prediction of ABO hemolytic disease of the
newborn.
Previous authors have indicated a racial difference
in the incidence of ABO hemolytic disease of the
newborn, with black infants being more frequently
affected than white infants. 2,8,9 Our data do not support
these findings, but the small number of black infants
in our series may have prevented our observation of
this association.
Because of the failure or prenatal tests, serologic
tests on cord blood may be the first useful tests avail-
able for predicting the clinical course of affected
infants, if a correlation can be shown to exist. Our
data seem to show such a relationship. First, infants
having positive direct antiglobulin test results have sig-
nificantly greater incidence and severity of jaundice
than do infants having laboratory evidence of erythro-
cytic sensitization (a positive heat eluate) and negative
direct antiglobulin test results. In fact, infants with nega-
tive direct antiglobulin test results do not show differ-
ences from the control infants, and thus either have sub-
clinical or compensated hemolytic disease or do not
have true hemolytic disease. Hence, it would appear that
the direct antiglobulin test has few, if any, false-
negatives results, making it a good screening test for
all newborn infants of group O mothers. Infants whose
direct antiglobulin test results are negative and control
infants appear to have the same likelihood of experi-
encing jaundice.
On the other hand, like most screening tests, the
direct antiglobulin test lacks specificity; that is, it is
not very useful for predicting severity of disease.
In our experience, and in that of others, 4,9 approxi-
373
mately 30% of infants having positive direct anti-
globulin test results will have more than "physiologic"
jaundice, with one-third to one-half of these requiring
therapy. A semi-quantitative test, the reaction strength
in a heat eluate of cord cells, may be of some value
in predicting severity of disease, since infants who had
tests showing stronger reactions had higher bilirubin
values and were more likely to require therapy, al-
though differences were not significant. These tests,
obviously, do not absolutely predict the course of the
infants, but may permit the laboratory to adivse the
clinician, who can closely monitor infants whose test
results indicate an unusual risk of jaundice.
Acknowledgment. Ms. Sondra Gandler Getz provided editorial
assistance.
References
1. American Association of Blood Banks: Technical Manual.
Seventh edition. Philadelphia, J. B. Lippincott Co., 1977,
pp 83-187
2. Bucher KA, et al: Racial difference in incidence of ABO
hemolytic disease. Am J Pub Health 66:854-858, 1976
3. Carapella-de Luca E, Pacioni C, Tonelli C: The titre of IgG
anti-A/B in the serum of group O mothers of incompatible
infants with early neonatal jaundice. Vox Sang 30:200-203,
1976
4. Clifford JH, et al: Screening for hemolytic disease of the
newborn by cord blood Coombs' testing-analysis of a five
year experience. Clin Pediatr 7:465-469, 1968
5. Gold ER, Butler NR: ABO hemolytic disease of the newborn.
tendon, John Wright and Sons, Ltd., 1972, pp 37-200
6. Graham H, Morrison M, McAndrew R: A simple method for
fne prediction of ABO incompatibility using Sephadex A-50.
Vox Sang 29:371-377, 1975
7. Halbrecht I: Role of hemagglutinins anti-A and anti-B in the
pathogenesis of jaundice of the newborn (icterus praecox
neonatorum). Am J Dis Child 68:248-249, 1944
8. Kaplan E, Herz F, Scheye E: ABO hemolytic disease of the
newborn without hyperbilirubinemia. Am J Hematol 1:279-
282, 1976
9. Kirkman HN: Further evidence for a racial difference in fre-
quency of ABO hemolytic disease. J Pediatr 90:717-721,
1977
10. Kochwa S, et al: Isoagglutinins associated with ABO erythro-
blastosis. J Clin Invest 40:874-882, 1961
11. Orzalesi M, et al: ABO system incompatibility: relationship
between direct Coombs' test positivity and neonatal jaundice.
Pediatrics 51:288-289, 1973
12. Rosenfield RE: A-B hemolytic disease of the newborn. Blood
10:17-28, 1955
13. Rosenfield RE, Ohno G: A-B hemolytic disease of the
newborn. Rev Hematol 10:231-235, 1955
14. Voak D, Bowley CC: A detailed serologic study on the pre-
diction and diagnosis of ABO hemolytic disease of the
newborn. Vox Sang 17:321-328, 1969