Rhesus Alloimmunization

Pathophysiology

The Rh complex is made up of a number of antigens, including C, D, E, c, d, e, and other variants, such as partial D antigens. More than 90% of cases of Rh alloimmunization are due to antibodies to the D antigen, and this is the only form of alloimmunization that can be prevented with Rh immune globulin prophylaxis. Therefore, this chapter is limited to a discussion of the D antigen, although the same principles apply to other antigen-antibody combinations. A person who lacks the D antigen on the surface of the red blood cells is regarded as being “RhD-negative,” and an individual with the D antigen is considered to be “RhD-positive.”

About 8% of African Americans are RhD-negative, whereas about 15% of white Americans are RhD-negative. Only 1-2% of Asian and 1-2% of Native Americans are RhD-negative. When RhD-negative patients are exposed to the RhD antigen, they may become sensitized. Most cases of sensitization are caused by a placental leak of fetal red blood cells into the maternal circulation (fetomaternal hemorrhage) during pregnancy. The fetal and maternal circulations are normally separated by the placental barrier. Small hemorrhages occur in either direction across the intact placenta throughout pregnancy. With advancing gestational age, the incidence and size of these transplacental (fetomaternal) hemorrhages increase, with the largest hemorrhages usually occurring at delivery. Most immunizations occur at the time of delivery, and antibodies appear either during the postpartum period or following exposure to the antigen in the next pregnancy.

Sensitization can also occur if an RhD-negative woman is exposed to RhD-positive blood via mismatched transfusion or hematopoietic stem cell transplantation or by injection with contaminated needles. In rare cases, the “grandmother” theory has been invoked. This theory suggests that an RhD-negative woman may have been sensitized from birth by receiving enough RhD-positive cells from her mother during her own delivery (i.e., a maternal-fetal hemorrhage) to produce an antibody response.

In general, two exposures to the RhD antigen are required to produce any significant sensitization, unless the first exposure is massive. The first exposure leads to primary sensitization, whereas the second causes an anamnestic response leading to the rapid production of immunoglobulins. The initial response to exposure to the RhD antigen is the production of immunoglobulin M (IgM) antibodies (which cannot cross the placenta) for a short period of time, followed by the production of IgG antibodies that are capable of crossing the placenta. If the fetus has the RhD antigen, these antibodies will coat the fetal red blood cells, causing them to be destroyed, or hemolyzed, in the spleen. If the hemolysis is mild, the fetus can compensate by increasing the rate of erythropoiesis. If the hemolysis is severe, it can lead to profound fetal anemia, resulting in extramedullary hematopoiesis, portal hypertension, hypoalbuminemia, hyperbilirubinemia, and heart failure (hydrops fetalis), as well as intrauterine fetal death. High bilirubin levels can damage the central nervous system and lead to neonatal encephalopathy and kernicterus. Before the widespread use of RhD immune globulin for prevention of RhD isoimmunization, kernicterus was one of the leading causes of cerebral palsy and sensorineural deafness.

If a pattern of mild, moderate, or severe disease has been established with two or more previous pregnancies, the disease tends either to be of the same severity or to become progressively more severe with subsequent pregnancies. If a woman has a history of fetal hydrops with a previous pregnancy, the risk of hydrops with a subsequent pregnancy is about 90%. Hydrops usually develops at the same time as or earlier than in the previous pregnancy.

Incidence

Although fetomaternal hemorrhage is very common, the incidence of RhD immunization within 6 months of the delivery of the first RhD-positive, ABO-compatible infant is only about 8%. In addition, the incidence of sensitization with the development of a secondary immune response before the next RhD-positive pregnancy is 8%. Therefore, the overall risk of immunization for the second full-term, RhD-positive, ABO-compatible pregnancy is about one in six pregnancies. The risk of RhD sensitization following an ABO-incompatible, RhD-positive pregnancy is only about 2%. The protection against immunization in ABO-incompatible pregnancies is due to the destruction of the ABO-incompatible cells in the maternal circulation and the removal of the red blood cell debris by the liver.

Fetomaternal hemorrhage may also occur before delivery. Establishment of the fetal circulation occurs at approximately 4 weeks' gestation, and the presence of the RhD antigen has been demonstrated as early as 38 days following conception. Consequently, RhD isoimmunization can occur at any time during pregnancy, from the early first trimester onward. In the first trimester, the most common causes of fetomaternal hemorrhage are spontaneous or induced abortions. The incidence of immunization following spontaneous abortion is 3.5%, whereas that following induced abortion is 5.5%. The risk is low in the first 8 weeks, but it rises to significant levels by 12 weeks' gestation. The risk of immunization following ectopic pregnancy is about 1%. Fetomaternal hemorrhage can also occur in the setting of second- or third-trimester vaginal bleeding, after invasive procedures such as amniocentesis or chorionic villus sampling, after abdominal trauma, or after external cephalic version. If necessary, the amount of fetal blood entering the maternal circulation after an episode associated with fetomaternal hemorrhage can be estimated using the Kleihauer-Betke test (described in the next section of this chapter). All pregnant RhD-negative women who are not sensitized to the D antigen should routinely receive prophylactic Rh immune globulin at 28 weeks' gestation, within 72 hours of delivery of an RhD-positive fetus, and at the time of recognition of any of the problems cited above that are associated with fetomaternal hemorrhage.

Detecting Fetomaternal Hemorrhage

The Kleihauer-Betke test is dependent on the fact that adult hemoglobin is more readily eluted through the cell membrane in the presence of acid than is fetal hemoglobin. The maternal blood is fixed on a slide with ethanol (80%) and treated with a citrate phosphate buffer to remove the adult hemoglobin. After staining with hematoxylin and eosin, the fetal cells can readily be distinguished from the maternal cells. All cells are then counted. The percentage of fetal cells present on the slide is determined and can be used to estimate the extent of the fetomaternal hemorrhage (measured in milliliters of whole blood) on the basis of the following equation:

As an example, if the Kleihauer-Betke is reported as 0.2%, then the estimated volume of fetal blood in the maternal circulation would be 0.002 × 5000, or 10 mL of fetal whole blood. There are a number of different formulas available for estimating the degree of fetomaternal hemorrhage, and all should be viewed as estimates based on their underlying assumptions regarding maternal and fetal blood volume. However, they are of value in determining the amount of Rh immune globulin to administer to prevent sensitization of an RhD-negative woman who is suspected of having a fetomaternal hemorrhage (refer to the “Prevention of RhD Alloimmunization” section later in this chapter).

Recognition of the At-Risk Pregnancy

A blood sample from every pregnant woman should be sent at the first prenatal visit for determination of the blood group and RhD type and for antibody screening. In RhD-negative patients whose anti-D antibody titers are positive (i.e., those who are RhD-sensitized), the RhD status of the father of the baby should be determined.