INTRODUCTION — RhD-negative patients who give birth to an RhD-positive newborn or who are otherwise exposed to RhD-positive red blood cells (RBCs) are at risk of developing anti-D antibodies. The RhD-positive fetuses/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. Where appropriate monitoring and intervention are available, HDFN can be treated successfully in most cases.
Implementation of programs for antenatal and postnatal anti-D immune globulin prophylaxis has led to a significant reduction in the frequency of D alloimmunization and associated fetal/neonatal complications. However, D alloimmunization with serious sequelae in offspring still occurs, particularly in resource-limited countries where anti-D immune globulin is not widely available .
This topic provides an overview of RhD alloimmunization in pregnancy. Prevention and management of this disorder, as well as management of pregnancies with alloimmunization to other red cell antigens, are reviewed in detail separately.
THE Rh SYSTEM — Obstetric providers should be aware of the different Rh (formerly called Rhesus) phenotypes and their clinical implications . The following discussion is a synopsis of key clinical issues related to the Rh blood group system. A detailed discussion of this 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 obstetric nomenclature for designating a pregnant individual's blood type is the ABO type and either "Rh positive" or "Rh negative." These terms are commonly used to describe an individual who has or does not have the D antigen on their red blood cells (RBCs). However, this abbreviated nomenclature is an artificial designation and can be confusing because the Rh blood group system consists of over 50 antigens ; the most common antigens that induce antibodies are D, C, c, E, and e. There is no d antigen, but C and c and E and e are alternate alleles with codominant expression.
Some combination of DCE is inherited as a haplotype from each parent. A pregnant person who is RhD negative can form anti-C, -c, -E, and/or -e antibodies if exposed to fetal RBCs with C, c, E, and/or e antigens inherited from the father that the mother does not share. Although an RhD-negative mother may have received prophylactic anti-D immune globulin, this would not prevent alloimmunization to other Rh antigens (c, C, E, e).
Prevalence of RhD-negative blood type — The prevalence of the D antigen varies among populations. The following examples illustrate the variation in prevalence of phenotypically RhD-negative individuals in different populations :
●Basques – 30 to 35 percent
●White North Americans or Europeans – 15 percent
●Black or African Americans – 8 percent
●Africans – 4 to 6 percent
●Indians – 5 percent
●Native Americans and Inuit people – 1 to 2 percent
●Japanese – 0.5 percent
●Thais – 0.3 percent
●Chinese – 0.3 percent
Zygosity — Approximately 40 percent of RhD-positive individuals are homozygous for the D antigen (DD); the remainder are heterozygous (Dd).
D variants — In White individuals, the primary molecular basis of the RhD-negative phenotype is absence of the D gene, RHD. In other phenotypically RhD-negative racial and ethnic groups, the D gene may be present but not translated or expressed .
Variants in the Rh system, particularly at the D and e loci, appear to be more common in populations of African descent. The RhD-negative phenotype in Africans is often the result of genes that contain sequences coding for D but do not produce the complete D antigen (ie, RHD pseudogene variant). The frequency of these gene distributions differs among Black African, African American, and Black South African individuals. An RHD pseudogene variant has been described in 69 percent of the Black South African population and 21 percent of the African American population .
Other unexpressed RHD gene variations have been observed in 12 percent of the Japanese population . These individuals have the entire RHD gene on their chromosomes but are RhD negative on serologic testing, and their RBCs are not affected by anti-D antibody in vivo.
Depending on the reagent employed, some individuals undergoing D typing will reveal a "weak D" expression on initial testing. When the individual's RBCs are tested with an anti-D reagent, they have no or less than a 2+ reaction; when antihuman globulin is added, a moderate to strong reaction occurs. These individuals are called "serologic weak D phenotype" (formerly "Du positive"). The two main explanations for this finding are either a decreased number of intact D antigens on the surface of the RBCs ("weak D type") or D antigens that are missing a portion, called epitopes ("partial D phenotype").
An estimated 0.2 to 1.0 percent of White individuals inherit RHD genes associated with the serologic weak D phenotype . Although more than 200 gene variants have been associated with alterations in the expression of the D antigen, the majority of weak D phenotypes (the proportion varies from 60 to 95 percent depending on the population) will be types 1, 2, or 3. Individuals with these three types who receive RhD-positive RBCs do not become alloimmunized to D. Other types of D variants can, however, form anti-D when exposed to the intact D antigen.
Problems arise when an individual has undergone testing in one laboratory using a sensitive reagent that detects the weak D phenotype and reveals an RhD-positive result but is subsequently tested in a second laboratory using a less sensitive reagent that reports an RhD-negative result. This may occur when the individual is initially tested as a blood donor and then is subsequently retested as part of routine prenatal care. In the United States, the Association for the Advancement of Blood & Biotherapies guideline for blood banks does not require performing a prenatal test for weak D or variant D, thus pregnant individuals with this blood type will be reported as RhD negative and should receive anti-D immune globulin, when indicated.
A working group with representatives from the blood banking and obstetric community recommends that when discrepant D typing is noted or a weak D type is detected, the individual should undergo RHD genotyping for weak D types. If they are type 1, 2, or 3, they can be considered RhD positive, and anti-D immune globulin is not indicated. This would avert the need of almost 25,000 units of anti-D immune globulin being administered to 13,000 pregnant individuals. In addition, more than 47,000 units of RhD-negative RBCs (a scarce commodity in the blood bank) would not be transfused to these patients when they require transfusion . If the individual is genotyped and weak D types 1, 2, or 3 are not found, then they are managed as RhD negative, and anti-D globulin is indicated. (See "RhD alloimmunization: Prevention in pregnant and postpartum patients", section on 'Weak D or discordant D typing'.)
G — A change of a single amino acid in the extramembranous portion of both the D and C proteins results in the expression of the G phenotype . The majority of individuals who are D and/or C positive are also G positive; if both D and C are absent, G is also absent. Individuals who lack all three antigens can become alloimmunized to the C and G antigen while not developing antibodies to D.
When an RhD-negative pregnant individual appears to have both anti-D and anti-C, the laboratory must determine whether the antibody is really anti-D or actually anti-G because a pregnant individual who develops anti-C and anti-G antibodies but no anti-D is still a candidate for anti-D immune globulin.
Anti-G is usually found in combination with anti-D, anti-C, or both, and thus, its significance in HDFN is difficult to quantify. Identification of anti-G can be accomplished through sequential adsorption/elution procedures. Anti-G (not anti-D and anti-C) should be suspected when the anti-C titer is greater than or equal to the anti-D titer. (See "RhD alloimmunization: Prevention in pregnant and postpartum patients".)
PATHOGENESIS OF ALLOIMMUNIZATION — By 38 days of gestation, the 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), D is only present on RBCs. Maternal D alloimmunization develops as a result of maternal immune system exposure to RhD-positive RBCs . Once anti-D immune globulin (Ig)G antibodies are present in the pregnant individual's circulation, they can cross the placenta and opsonize fetal RBCs, which are then phagocytized by macrophages in the fetal spleen, resulting in fetal anemia.
Events that can cause maternal alloimmunization include:
●Transplacental fetomaternal bleeding during any pregnancy
●Injection with needles contaminated by RhD-positive blood [13-15]
●Inadvertent transfusion of RhD-positive blood
●RhD-mismatched allogeneic hematopoietic stem cell transplantation 
Transplacental fetomaternal bleeding accounts for virtually all cases of maternal D alloimmunization. Tiny (0.1 mL) quantities of fetal RBCs spontaneously gain access to the maternal circulation in nearly all pregnancies, as demonstrated by studies using flow cytometry . The frequency and volume of spontaneous fetomaternal bleeding increase with advancing gestational age and are highest at delivery . Fetomaternal bleeding can also be associated with events such as miscarriage, pregnancy termination, ectopic pregnancy, invasive in-utero procedures, fetal death, maternal abdominal trauma, antepartum maternal hemorrhage, and external cephalic version. Early predictors of fetomaternal bleeding remain largely unknown, and no cause can be identified in over 80 percent of cases .
Of note, there are at least six reports of alloantibodies to the D antigen without identifiable maternal exposure to RBCs carrying the D antigen . 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, RBCs from the individual's heterozygous RhD-positive mother gain access to the fetal circulation at birth (maternal-fetal hemorrhage). The RhD-negative neonate then forms a low level of antibody to these RhD-positive cells.
Although the D antigen is thought to elicit a strong immune response, the response varies considerably among individuals. As an example, studies in RhD-negative incarcerated males showed that intravenous injection of as little as 0.1 mL of RhD-positive RBCs was sufficient to immunize some individuals, while 30 percent of the RhD-negative males did not become sensitized despite two injections (first 10 mL, then 5 mL) of RhD-positive blood over a six-month period [21,22].
The percentage of RhD-negative individuals developing an immune response to infusion of RhD-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) . The antibody response develops slowly and is usually not detectable serologically until 5 to 15 weeks after exposure.
Whether a primary immune response occurs also depends upon several factors besides the volume of fetal blood to which the mother was exposed. These variables include the frequency of fetomaternal bleeding and whether the mother and fetus are ABO compatible . Both the immunogenicity of the fetal RBCs and the immunogenic response capacity of the mother play a role in pathogenesis. For example, individuals with acquired immune deficiency syndrome (AIDS), transplant patients on immunosuppressive medication, trauma victims, and cancer patients on intensive chemotherapy may not form alloantibodies to the D antigen .
FETAL/NEONATAL CONSEQUENCES OF ALLOIMMUNIZATION
●Hemolytic disease of the fetus and newborn (HDFN) – Transplacental transfer of maternal antibody leads to HDFN. 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 that affect Fc receptor function; and human leukocyte antigen (HLA)-related inhibitory antibodies . (See "Alloimmune hemolytic disease of the newborn: Postnatal diagnosis and management".)
●Hydrops fetalis – Hydrops fetalis (two or more of the following: skin edema, ascites, pericardial effusion, pleural effusion) in HDFN occurs when the fetal hemoglobin deficit is at least 7 g/dL below the mean for gestational age (consistent with a hematocrit less than approximately 15 percent or hemoglobin <5 g/dL) .
●Thrombocytopenia and neutropenia – HDFN may also be associated with thrombocytopenia and neutropenia . The risk increases with increasingly severe anemia and is most common in hydropic fetuses . In a systematic review, 11 to 26 percent of fetuses undergoing initial umbilical vein sampling because of D alloimmunization had platelet counts <150,000/microL . However, severe thrombocytopenia (<50,000/microL) is uncommon (prevalence 18 percent in one study). The mechanism of thrombocytopenia may be decreased production due to increased production of red blood cells (RBCs), but increased consumption or destruction of platelets has also been hypothesized.
SCREENING — D typing and an antibody screen should be performed at the first prenatal visit. For RhD-negative individuals with an initially negative antibody screen and uncomplicated pregnancy, the antibody screen is repeated at approximately 28 weeks of gestation and at admission for labor and delivery [30,31]. Screening can be performed with saline tube methods or less commonly by gel microcolumn. If the antibody screen is positive, the antibody titer is determined.
●Indirect Coombs – The indirect Coombs test is the most commonly used method for determining the titer. Incubation of known RhD-positive red blood cells (RBCs) with maternal plasma is the first step. Any anti-D antibody present will adhere to the RBCs, which are then washed and suspended in antihuman globulin (Coombs) serum. RBCs coated with maternal anti-D will be agglutinated by the antihuman globulin, which is referred to as a positive indirect Coombs test. The titer is the highest dilution at which agglutination occurs (eg, a 1:16 titer means the patient's plasma is positive at any dilution down to 1 part plasma to 15 parts diluent). Tube dilutions are set up to report titers of one to: 1, 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, 2048, and 4096. Thus, for a patient with a titer of 1:4, a titer of 1:8 would represent a one tube increase and a titer of 1:16 would represent a two tube increase in titer.
●Gel microcolumn assay (GMA) – The GMA card is used by an increasing number of blood banks to replace the traditional tube agglutination tests for identifying anti-D antibody and determining the titer. Advantages of GMA over tube tests include increased sensitivity, less susceptibility to interlaboratory and intralaboratory sources of variability, clear objective results that are stable, less time consuming, and compatible with automation . A major disadvantage is that a critical titer threshold has not been defined for gel method. Large prospective studies are needed to provide validation data correlating GMA results and tube titrations using a consistent technique and correlating GMA results and neonatal outcomes.
Although not the primary test used for screening, the indirect Coombs titer (standard tube titer) is the value that should be used to guide obstetric management of alloimmunized pregnancies. GMA usually yields higher titer values than tube tests [33,34]. In one review of 384 paired samples from eight studies, GMA results were 1.4 to 2 dilutions above tube results . The authors suggested that if a tube titer of 16 is considered critical, then a reasonable critical titer using GMA would be 64 because a two tube enhanced value is most common. However, they noted some studies reported GMA results as much as 5 additional dilutions above tube results and one reported GMA results were similar to tube results. Clinicians should therefore consult with their laboratory to determine the methodology used for antibody titration.
DIAGNOSIS — The diagnosis of D alloimmunization is based upon detection of anti-D antibody in maternal blood. Identification of an alloantibody means that the fetus is at risk for hemolytic disease of the fetus and newborn (HDFN), not that it has occurred or will develop.
False positive — Screening for anti-D antibodies may not be helpful in identifying alloimmunization if the patient has received anti-D immune globulin within the past few weeks. Titration can be helpful in these cases: individuals who received anti-D immune globulin at 28 weeks will have a low (0 to ≤4) antibody titer at term; a titer >4 suggests alloimmunization. Also, new alloimmunization is associated with IgM antibodies, whereas exogenous anti-D is IgG.
MANAGEMENT — Management of pregnancies complicated by maternal alloimmunization involves the following principles , all of which are discussed in detail separately. (See "RhD alloimmunization in pregnancy: Management".)
●Determine the fetal RhD type. The fetal RhD type may be determined by testing fetal DNA in maternal serum, or it may be inferred if quantitative polymerase chain reaction (PCR) to identify the number of paternal RHD genes reveals homozygosity and paternity is assured.
●Monitor for fetal anemia if the fetus is RhD positive. Monitoring depends on the clinical scenario and may involve following maternal anti-D titers in conjunction with ultrasound assessment of fetal middle cerebral artery peak systolic velocity (MCA-PSV).
Performing serial combined maternal plasmapheresis and intravenous immune globulin therapy is a promising approach for decreasing the severity of fetal disease when there is a significantly elevated titer (eg, >1024) or a history of previous severe hemolytic disease of the fetus and newborn (HDFN; eg, fetal or neonatal death due to HDFN or need for intrauterine transfusion [IUT] before 24 weeks of gestation) .
●If severe anemia is suspected on MCA-PSV, it should be confirmed by fetal blood sampling to determine the fetal hemoglobin/hematocrit level.
●Treat severe fetal anemia near term by delivery for neonatal treatment; remote from term (<35 weeks gestation), perform intrauterine fetal transfusions.
NEWBORN OUTCOME — 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 RhD-positive newborns of mothers with RhD alloimmunization underwent exchange transfusion .
If the neonatal hematocrit is near normal because of a recent intrauterine transfusion (IUT), neonatal exchange transfusion may not be necessary. Serial bilirubin measurements with phototherapy when the phototherapy threshold is met are still warranted to prevent kernicterus . The use of intravenous immune globulin (IVIG) is controversial and may be harmful [39-41].
With senescence of the transfused red cells, approximately 60 to 70 percent of newborns transfused in utero will require a "top-up" transfusion at one month of age due to suppression of fetal erythropoiesis from IUT and persistence of maternal antibody not removed by exchange transfusion. In these cases, a median of two top-up transfusions is necessary before reticulocytosis begins and maternally acquired anti-red blood cell antibodies disappear .
A detailed discussion of the evaluation and management of neonates of alloimmunized mothers is available elsewhere. (See "Alloimmune hemolytic disease of the newborn: Postnatal diagnosis and management".)
PREVENTION — Most, but not all, D alloimmunization can be prevented by administration of anti-D immune globulin to females exposed or at high risk of being exposed to RhD-positive red blood cells (RBCs). (See "RhD alloimmunization: Prevention in pregnant and postpartum patients".)
Once alloimmunization has occurred, anti-D immune globulin is not effective for preventing or reducing the severity of hemolytic disease of the fetus and newborn (HDFN). The three options for prevention of an affected fetus in this setting are:
●Pregnancy by insemination with sperm from an RhD-negative donor
●In vitro fertilization and preimplantation genetic testing for selection of RHD-negative embryos (if father is heterozygous for RHD)
●Use of a gestational carrier
Given the success of intrauterine fetal transfusion for treatment of HDFN, one of these options is usually considered only by individuals at risk for very early, severe fetal anemia in whom intrauterine fetal transfusion is more risky and less successful. (See "Donor insemination" and "Preimplantation genetic testing" and "Gestational carrier pregnancy".)
ALLOIMMUNIZATION OTHER THAN D — Diagnosis and management of pregnant individuals with alloimmunization to non-D red blood cell (RBC) antigens are reviewed separately . (See "Management of non-RhD red blood cell alloantibodies during pregnancy".)
SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Rh disease in pregnancy".)
INFORMATION FOR PATIENTS — UpToDate offers two types of patient education materials, "The Basics" and "Beyond the Basics." The Basics patient education pieces are written in plain language, at the 5th to 6th grade reading level, and they answer the four or five key questions a patient might have about a given condition. These articles are best for patients who want a general overview and who prefer short, easy-to-read materials. Beyond the Basics patient education pieces are longer, more sophisticated, and more detailed. These articles are written at the 10th to 12th grade reading level and are best for patients who want in-depth information and are comfortable with some medical jargon.
Here are the patient education articles that are relevant to this topic. We encourage you to print or e-mail these topics to your patients. (You can also locate patient education articles on a variety of subjects by searching on "patient info" and the keyword(s) of interest.)
●Basics topics (see "Patient education: Pregnancy in Rh-negative people (The Basics)")
SUMMARY AND RECOMMENDATIONS
●Rh system – The 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 Rh system' above.)
●Typing and screening – RhD typing and an antibody screen are routine performed at the first prenatal visit. In RhD-negative individuals, the antibody screen should be repeated at 28 weeks of gestation and again at delivery. (See 'Screening' above.)
•When discrepant RhD typing is noted or a weak D type is detected, the patient should undergo RHD genotyping for weak D types. If they are type 1, 2 or 3, they can be considered RhD positive and anti-D immune globulin is not indicated (see 'D variants' above). If genotype testing is not available, the patient should be treated as if they are RhD negative and administered anti-D immune globulin for standard indications.
●Diagnosis – The diagnosis of D alloimmunization is based upon detection of anti-D antibody in maternal serum. (See 'Diagnosis' above.)
●Pathogenesis of alloimmunization – Maternal D alloimmunization develops as a result of maternal immune system exposure to RhD-positive red blood cells (RBCs). Maternal immunization can occur as a result of transplacental fetomaternal hemorrhage during any pregnancy (table 1), injection with needles contaminated by RhD-positive blood, or inadvertent transfusion of RhD-positive blood (including during transplantation). (See 'Pathogenesis of alloimmunization' above.)
●Consequences – Transplacental transfer of maternal antibody leads to hemolytic disease of the fetus and newborn (HDFN). The severity of fetal anemia is influenced primarily by antibody concentration. Hydrops fetalis in HDFN occurs when the fetal hemoglobin deficit is at least 7 g/dL below the mean for gestational age. (See 'Fetal/neonatal consequences of alloimmunization' above.)
●Management – Management of pregnancies complicated by maternal alloimmunization involves (see 'Management' above):
•Determining the fetal RhD type by testing fetal DNA in maternal serum, or it may be inferred if quantitative polymerase chain reaction (PCR) to identify the number of paternal RHD genes reveals homozygosity and paternity is assured.
•Monitoring for fetal anemia if the fetus is RhD positive. Monitoring may involve following maternal anti-D titers in conjunction with ultrasound assessment of fetal middle cerebral artery peak systolic velocity (MCA-PSV).
•Treating severe fetal anemia. Near term, the fetus is delivered for neonatal treatment; remote from term, intrauterine fetal transfusions are performed. Exchange transfusions may be required after birth. (See 'Newborn outcome' above.)
●Prevention – Most, but not all, D alloimmunization can be prevented by administration of anti-D immune globulin to females exposed or at high risk of being exposed to RhD-positive RBCs. (See 'Prevention' above.)
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