Overview of Rhesus D alloimmunization in pregnancy

Author
Kenneth J Moise Jr, MD
Section Editor
Charles J Lockwood, MD, MHCM
Deputy Editor
Vanessa A Barss, MD, FACOG
Contributor disclosures
All topics are updated as new evidence becomes available and our peer review process is
complete.
Literature review current through: May 2016. | This topic last updated: Aug 24, 2015.
INTRODUCTION Rhesus (Rh)-D negative women who deliver an Rh(D) positive baby or who
are otherwise exposed to Rh(D) positive red cells are at risk of developing anti-D antibodies.
Rh(D) positivefetuses/neonates of these mothers are at risk of developing hemolytic disease of
the fetus and newborn (HDFN), which can be associated with serious morbidity or mortality.
Implementation of programs for antenatal and postnatal anti-D immune globulin prophylaxis has
led to a significant reduction in the frequency of Rh(D) alloimmunization and
associated fetal/neonatalcomplications. However, Rh(D) alloimmunization with serious sequelae
in offspring still occurs, particularly in low resource countries where anti-D immune globulin is
not widely available [1]. Where appropriate monitoring and intervention are available, HDFN can
be treated successfully in most cases.
THE RHESUS SYSTEM Obstetricians should be aware of the different Rh(D) phenotypes
and their clinical implications [2]. The following discussion is a synopsis of key clinical issues
related to the Rhesus system. A detailed discussion of the Rhesus system, including its variants,
can be found separately. (See "Red blood cell antigens and antibodies", section on 'Rh blood
group system'.)
D, d, C, c, E, e, and G The standard obstetrical nomenclature for designating a pregnant
womans blood type is the ABO type and either Rh positive or Rh negative. These terms are
commonly used to describe a woman who has or does not have the Rh(D) antigen on her red
cells (RBCs). However, this abbreviated nomenclature is not technically correct because the
Rhesus system consists of numerous other antigens, most commonly C, c, E, e, and G (there is
no d antigen). Thus, a woman who is "Rh-negative" (meaning no D antigen) but carries the C
antigen on her RBCs can develop anti-c antibodies as a result of transplacental bleeding of c
positive fetal RBCs. Since she is Rh(D) negative, she may have received prophylactic antiDimmune globulin in previous pregnancies, but this would not prevent c alloimmunization.
Prevalence of Rh(D)-negative blood type The prevalence of Rhesus antigens varies
among populations. The following examples illustrate ethnic variation in prevalence of
phenotypically Rh(D)-negative individuals [3]:
Basques 30 to 35 percent

Caucasians in North America and Europe 15 percent

African Americans 8 percent
Africa 4 to 6 percent
India 5 percent
Native Americans and Inuit Eskimos 1 to 2 percent
Japan 0.5 percent
Thailand 0.3 percent
China 0.3 percent
Zygosity About 40 percent of Rh(D)-positive individuals are homozygous for the D antigen
(DD); the remainder is heterozygous (Dd).
Rh(D) variants In Caucasians, the primary molecular basis of the Rh(D)-negative phenotype
is absence of the D gene (RHD). In other phenotypically Rh(D)-negative ethnic groups, the gene
may be present but not translated or expressed, weakly expressed, or partially expressed [4].
These Rh(D) antigen variants, variously called partial D or D mosaic (one or more epitopes of
the antigen are absent), weak D (epitopes of the antigen are weakly expressed), Rh mod, and
D(el) may type as Rh(D)-negative or Rh(D)-positive by serologic reagents [4-8]. Patients with
these variants are potentially at risk of developing anti-D antibody, and their RBCs may induce
anti-D antibody formation in Rh(D)-negative recipients.
Variants in the Rhesus system appear to be more common in the African-American population,
particularly at the D and e loci. The Rh(D)-negative phenotype in Africans is often the result of
genes that contain sequences coding for Rh(D) but do not produce the complete Rh(D) antigen
(ie, RHD pseudogene). The frequency of these gene distributions differs between
black Africans, African Americans, and black South Africans. An RHD pseudogene has been
described in 69 percent of South African blacks and 21 percent of African Americans [9]. By
comparison, only 0.96 percent of individuals in a Canadian study had a weak Rh(D) response
when serotyped [7]. Most of these individuals express a reduced number of Rh(D) antigens on
the surface of their red cells, although a few have a partial D antigen (also known as D variant)
[10]. Since the majority of these individuals express the Rh(D) antigen, they rarely form anti-D
antibody. However, women with partial D antigen may develop antibodies against D epitopes
absent on maternal red cells but present on fetal red cells.
In the United States, the AABB (formerly the American Association of Blood Banks) guideline for
blood banks does not require that a test for weak D or variant D be performed on Rh(D)negative pregnant patients. However, some commercial laboratories perform prenatal blood
typing using reagents that will detect the weak D or variant D phenotype, and thus report this
result when present. More commonly, a monoclonal typing sera is used for Rh(D) typing that
does not detect weak D or D variant phenotypes so these patients are reported as Rh(D)negative and will be candidates for anti-D immune globulin.

Of note, women with weak D or partial D who donate blood will undergo indirect antiglobulin
testing and will be reported as Rh(D)-positive to ensure that their blood is not transfused into an
Rh(D)-negative recipient who could then form anti-D antibodies. The different blood typing
policies for blood donors versus pregnant women and transfusion recipients result in some
women being Rh(D)-positive in one setting and Rh(D)-negative in the other setting.
Other unexpressed RHD gene variations have been observed in 12 percent of Japanese
individuals [11]. These individuals have the entire RHD gene on their chromosomes, but are
Rh(D)-negative on serologic testing and their red cells are not affected by anti-D antibody in
vivo.
Rh(G) A change of a single amino acid in the extramembranous portion of both the Rh(D)
and Rh(C) proteins results in the expression of the Rh(G) phenotype [12]. The majority of
patients who are Rh(D)and/or Rh(C) positive are also Rh(G) positive; if both Rh(D) and Rh(C)
are absent, Rh(G) is also absent. Patients who lack all three antigens can become
alloimmunized to the Rh(C) and Rh(G) antigen while not developing antibodies to Rh(D). When
an Rh(D)-negative pregnant woman appears to have both anti-D and anti-C at a similar level of
titer, the laboratory must determine whether the antibody is really anti-D or actually anti-G
because a pregnant woman who develops anti-C and anti-G antibodies but no anti-D is still a
candidate for anti-D immune globulin. (See "Prevention of Rh(D) alloimmunization in
pregnancy".)
PATHOGENESIS AND CONSEQUENCES OF ALLOIMMUNIZATION By 30 days of
gestation, the Rh(D) antigen is expressed as part of the red blood cell (RBC) membrane, and, in
contrast to most other antigens (eg, A,B,M,N), Rh(D) is only present on RBCs. Maternal Rh(D)
alloimmunization develops as a result of maternal immune system exposure to Rh(D)-positive
RBCs [13]. Once anti-D IgG antibodies are present in the pregnant woman's circulation, they
can cross the placenta and opsonize fetal RBCs, which are then phagocytized by macrophages
in the fetal spleen.
Events that can cause maternal alloimmunization include:
Transplacental fetomaternal hemorrhage during any pregnancy (table 1)
Injection with needles contaminated by Rh(D)-positive blood [14]
Inadvertent transfusion of Rh(D)-positive blood
D-mismatched allogeneic hematopoietic stem cell transplantation [15]
Transplacental fetomaternal bleeding accounts for virtually all cases of maternal Rh(D)
alloimmunization. Tiny (0.1 mL) quantities of fetal RBCs gain access to the maternal circulation
in nearly all pregnancies, as demonstrated by studies using flow cytometry [16]. The frequency
and volume of spontaneous fetomaternal hemorrhage increase with advancing gestational age
and are highest at delivery [17]. Fetomaternal hemorrhage can also be associated with
miscarriage, pregnancy termination, ectopic pregnancy, invasive in-utero procedures, fetal
death, maternal abdominal trauma, antepartum maternal hemorrhage, and external cephalic
version.

Of note, there are six reports of alloantibodies to Rh(D) antigen without identifiable maternal
exposure to red cells carrying the D antigen [18]. These cases may be the result of early
pregnancy losses (including vanishing twins) that were not clinically recognized. Alternatively, a
"grandmother theory" has been proposed as the etiology. In these cases, Rh(D) positive red
cells from the patient's mother gain access to the fetal circulation at birth (maternal-fetal
hemorrhage). The Rh(D) negative neonate then forms a low level of antibody to these Rh(D)
positive cells.
Although the Rh(D) antigen is thought to elicit a strong immune response, the response varies
considerably among individuals. As an example, studies in male Rh(D)-negative prisoners
showed that intravenous injection of as little as 0.1 mL of Rh(D)-positive RBCs was sufficient to
immunize some individuals, while 30 percent of the Rh(D)-negative men did not become
sensitized despite two injections (first 10 mL, then 5 mL) of Rh(D)-positive blood over a sixmonth period [19,20].
The percentage of Rh(D)-negative individuals developing an immune response to infusion of
Rh(D)-positive RBCs depends, in part, on the volume of blood infused: 0.5 mL RBCs stimulates
an anti-D response in some subjects, whereas one unit (450 mL) of RBCs results in the
maximum percentage of responders (80 percent) [21]. The antibody response develops slowly,
and is usually not detectable serologically until 5 to 15 weeks after exposure. Whether a primary
immune response occurs depends upon several factors besides the volume of fetal blood to
which the mother was exposed. These variables include the frequency of fetomaternal
transfusion and whether the mother and fetus are ABO compatible [13]. Both the
immunogenicity of the fetal RBCs and the immunogenic response capacity of the mother play a
role in pathogenesis. Interestingly, individuals with acquired immune deficiency syndrome
(AIDS) may not form alloantibodies to the D antigen [22].
Transplacental transfer of maternal antibody leads to hemolytic disease of the fetus/newborn.
The severity of fetal anemia is influenced primarily by antibody concentration, but also by
additional factors that are not fully understood. These include the subclass and glycosylation of
maternal antibodies; the structure, site density, maturational development and tissue distribution
of blood group antigens; the efficiency of transplacental IgG transport; the functional maturity of
the fetal spleen; polymorphisms which affect Fc receptor function; and the presence of human
leukocyte antigen (HLA)-related inhibitory antibodies [23]. (See "Postnatal diagnosis and
management of hemolytic disease of the fetus and newborn".)
Severe anemia leads to hydrops fetalis (two or more of the following: skin edema, ascites,
pericardial effusion, pleural effusion). Hydrops occurs when the fetal hemoglobin deficit is at
least 7 g/dL below the mean for gestational age (consistent with a hematocrit less than about 15
percent or hemoglobin <5g/dL)[24].
DIAGNOSIS The diagnosis of Rh(D) alloimmunization is based upon detection of anti-Rh(D)
antibody in maternal serum.
SCREENING Rh(D) typing and an antibody screen should be performed at the first prenatal
visit. For Rh(D)-negative women with an initially negative antibody screen and uncomplicated
pregnancy, the antibody screen is repeated at 28 weeks of gestation, and at delivery [25,26]. A
positive antibody test should be interpreted as a screening test, just as serum alpha-fetoprotein

is a screening test for the detection of fetal neural tube defects. A positive screening test means
that the fetus is at risk for hemolytic disease, not that it has occurred or will develop.
The test most commonly used for diagnostic purposes is the indirect Coombs test (ie,
determination of antibodies in the plasma), which is the most accurate technique for determining
antibody titers. The indirect Coombs titer is the value used to guide obstetrical management of
alloimmunized pregnancies.
Incubation of known Rh(D)-positive red blood cells (RBCs) with maternal plasma is the first step
in the indirect Coombs test. Any anti-Rh(D) antibody present will adhere to the RBCs. The RBCs
are then washed and suspended in antihuman globulin (Coombs) serum. Red cells coated with
maternal anti-Rh(D) will be agglutinated by the antihuman globulin, which is referred to as a
positive indirect Coombs test.
The gel microcolumn assay (GMA) card is a promising alternative to traditional tube
agglutination tests for determining anti-D antibody titers. Advantages of GMA over tube tests
include: it is less susceptible to inter-laboratory and intra-laboratory sources of variability, yields
clear objective results that are stable, takes less time, and is compatible with automation [27].
However, GMA may yield higher titer values compared to tube tests. In several studies, the
increase was usually only one or two dilutions, which is not clinically significant (a four-fold
difference is clinically significant) [27,28], but some studies reported greater discordancy
[29,30]. More data are needed to establish the correlation between GMA titer and severity of
fetal anemia before this assay can be used to manage alloimmunization in pregnancy. For this
reason, standard tube titers should still be used for clinical management of the alloimmunized
pregnancy.
Use of automated enzymatic methods for screening for anti-Rh(D) antibodies is not
recommended. These techniques are less accurate than manual methods and, in rare cases,
detect clinically insignificant, naturally occurring Rh(D) antibodies [31].
MANAGEMENT OF PREGNANCIES COMPLICATED BY
ALLOIMMUNIZATION Management of pregnancies complicated by maternal
alloimmunization involves determining the fetal Rh(D) type and monitoring for fetal anemia if the
fetus is Rh(D) positive [32]. Monitoring may involve following maternal anti-D titers or ultrasound
assessment of fetal middle cerebral artery peak systolic velocity. Severe fetal anemia near term
is treated by delivery for neonatal treatment; remote from term, intrauterine fetal transfusions
are performed. Serial combined maternal plasmapheresis and intravenous immune
globulintherapy is a promising approach for decreasing the severity of fetal disease, but is
investigational. These subjects are reviewed in detail separately. (See "Management of
pregnancy complicated by Rhesus (D) alloimmunization".)
NEONATAL ISSUES In a report of hemolytic disease of the newborn detected, managed and
treated by the Regional Blood Transfusion Center of France over a 30 year interval, 62 percent
of Rh(D)-positive newborns of women with Rh(D)-alloimmunization underwent exchange
transfusion [33]. A detailed discussion of the evaluation and management of neonates of
alloimmunized women is available elsewhere. (See "Postnatal diagnosis and management of
hemolytic disease of the fetus and newborn".)

PREVENTION Most, but not all, Rh(D) alloimmunization can be prevented by administration
of anti-Dimmune globulin to women exposed or at high risk of being exposed to Rh(D)-positive
red blood cells (RBCs). (See "Prevention of Rh(D) alloimmunization in pregnancy".)
Once alloimmunization has occurred, anti-D immune globulin is not effective for preventing or
reducing the severity of fetal/neonatal disease. Alloimmune fetal/neonatal anemia can be
treated, but the only options for prevention of the disease are pregnancy by insemination with
sperm from a Rh(D)-negative donor, preimplantation genetic diagnosis for selection
of RHD negative embryos (if father is heterozygous forRHD), or use of a gestational surrogate.
Given the success of intrauterine fetal transfusion, these options are usually considered only by
women at risk for very early, severe fetal anemia where intrauterine fetal transfusion is more
risky and less successful. (See "Donor insemination" and "Preimplantation genetic
diagnosis" and "Surrogate pregnancy".)
ALLOIMMUNIZATION OTHER THAN RH(D) Diagnosis and management of pregnant
women with alloimmunization to non Rhesus red blood cell (RBC) antigens are reviewed
separately [34]. (See"Management of non-Rh(D) red blood cell alloantibodies during
pregnancy".)
SUMMARY AND RECOMMENDATIONS
The Rhesus (Rh) blood system consists of the C, c, D, E, e, and G antigens (there is no
d antigen). Variants include weak D and partial D antigens. In addition, the D gene may be
present, but not translated or not expressed. (See 'The Rhesus system' above.)
Maternal Rh(D) alloimmunization develops as a result of maternal immune system
exposure to Rh(D)-positive red blood cells (RBCs). Maternal immunization can occur as a
result of transplacental fetomaternal hemorrhage during any pregnancy, injection with
needles contaminated by Rh(D)-positive blood, or inadvertent transfusion of Rh(D)-positive
blood (including during transplantation). (See 'Pathogenesis and consequences of
alloimmunization' above.)
The diagnosis of Rh(D) alloimmunization is based upon detection of anti-Rh(D) antibody
in maternal serum. (See 'Diagnosis' above.)
We recommend performing Rh(D) typing and an antibody screen at the first prenatal visit
(Grade 1B). In Rh(D)-negative women, the antibody screen may be repeated at 28 weeks
of gestation, and should be repeated at delivery. (See 'Screening' above.)