Clinical Perspectives: Rh Immunoglobulin and Hemolytic Disease of the Fetus and Newborn

In these modern times, it can sometimes be difficult to recognize the significant impact that many medications have had in eliminating fatal conditions and diseases. For any woman who has been pregnant and has the Rh-Negative blood group, this medication is Rh Immune Globulin (or RhIg for short).


WinRho, the Rh Immunoglobulin medication used at my hospital

What role does RhIg play in the pre-natal and post-partum care of mothers and their babies? To explain, I must first discuss one of the most important blood groups after the ABO blood group, known as the Rh blood group. This blood group has a very strong immune response, with the main antigen known as the D antigen. The presence or absence of the D antigen determines the “Positive” or “Negative” designation of your blood group. For example, I am B Positive, meaning I have the B antigen of the ABO blood group and the D antigen of the Rh blood group.

Historically, the Rh blood group was named as such because when scientists Karl Landsteiner and Alexander S. Wiener first discovered the D antigen, they thought it was similar to an antigen found in rhesus monkeys. Even though it was later found out that this was not the case, the name stuck. This blood group was unfortunately discovered due to the untimely demise of a fetus, a disease now known as hemolytic disease and the fetus and newborn (HDFN). HDFN occurs whenever there is an antigenic response from the mother against the fetus’ blood. During pregnancy and at birth, maternal and fetal blood have the opportunity to come into contact with each other. For mothers who are typed as Rh-Negative and give birth to children who are typed as Rh-Positive, the D antigens present in the fetal blood can stimulate an immune response, causing the mother to produce an antibody known as anti-D. This antibody is an IgG antibody that upon first exposure to the antigen, takes 120 days to appear in the immune system. Therefore, during the first exposure of the D antigen, which usually occurs at the birth of the first Rh-Positive child, the child remains unharmed from the antibody. However, in subsequent exposures of the D antigen with the mother’s blood, the anti-D is produced much more quickly at two to seven days. Due to the small size of the IgG antibody, it is able to cross the placental wall, bind to fetal red blood cells, and cause destruction of fetal red blood cells by lysis. While most fetal-maternal blood exposure occurs during delivery, there are other circumstances, such as miscarriage, abdominal trauma during pregnancy, amniocentesis, and more, that can also put Rh-Negative mothers at risk of producing anti-D.


Representation of Rh-Negative woman (-) carrying Rh-Positive fetus (+). Once fetal blood enters the maternal circulation, anti-D (triangles) are produced and can bind to fetal cells.

What exactly occurs when the anti-D attaches to Rh-Positive fetal cells? First, the cells are removed by white blood cells known as macrophages, and are transported to the liver and spleen. Due to the removal of these cells, anemia develops, and the bone marrow compensates by producing immature red blood cells. When severe anemia occurs, hydrops fetalis develops which presents with edema, effusions, and ascites due to an enlarged liver and spleen. In addition, increased levels of unconjugated bilirubin are present due to red blood cell destruction. While the fetus is being carried by the mother, it is protected by the mother’s enzyme, glucuronyl transferase, which conjugates the bilirubin to a water-soluble form that is excreted in the urine. Since newborns lack this enzyme, after delivery from the mother they are unable to conjugate the bilirubin and it builds up in lipid-rich areas of the brain, resulting in a condition known as kernicterus. Bilirubin is oxidized by light; therefore, phototherapy is used to treat kernicterus and prevent brain damage. An exchange transfusion is also performed to improve the newborn’s anemia, remove unconjugated bilitubin, and decrease the amount of maternal anti-D present in the newborn’s blood. During this transfusion, the newborn’s blood is being replaced with compatible blood. Blood transfused into newborns must be no more than seven days old, Rh-Negative, ABO group specific, antigen negative for any maternal antibodies, negative for cytomegalovirus, irradiated, and sickle cell negative.


Newborn under ultraviolet light (phototherapy) to conjugate high levels of unconjugated bilirubin in blood

How does one prevent HDFN from occurring? Pre-natal laboratory testing can be performed on mothers in order to prevent HDFN. First, an ABO group and Rh type is determined. In addition, an antibody screen is performed to determine if any clinically significant antibodies are present. For women who are Rh-Negative and have a negative antibody screen, the woman is eligible to receive RhIg to prevent the occurrence of HDFN. RhIg is given at two separate occasions: first at 28 weeks gestation, and second within 72 hours post-delivery. For any Rh-Positive fetal cells that are present in the maternal circulation, the RhIg will bind to them and they will be removed to the spleen, inhibiting the mother’s immune response from producing anti-D. Cases of women producing maternal anti-D has decreased substantially over the years with the pre-natal and post-partum doses of RhIg, however, if an antibody screen is positive for anti-D, the pregnancy must be monitored by performing antibody titres every four weeks. An antibody titre measures the dilution at which the antibody is still reactive. A comparison is made against the current titre and the past titre to see if a significant difference in titre has occurred.


ABO grouping and Rh type by tube method showing A Positive blood group. This is the main method I use to perform blood groups in the transfusion medicine laboratory.

Newborns are also tested for their ABO group and Rh type at birth, as well for the direct antiglobulin test (DAT). The DAT determines whether there is any antibody coating the newborn’s red blood cells, such as maternal anti-D. The Rh type of the newborn also determines whether the mother receives any RhIg post-delivery, as Rh-Negative women who have Rh-Negative babies will not produce anti-D and do not require RhIg after birth. However, for babies who initially give a weak or negative reaction to Rh typing, a “Weak D” test is performed, in which the baby’s test sample is incubated at 37⁰C for 15 minutes, washed three times, and has commercial anti-IgG added to it. This additional test is completed in order to determine the newborn’s true Rh status and whether the mother should receive RhIg after birth. However, if the baby’s DAT is positive and their Rh typing appears negative at first, this may be a false negative reaction due to the newborn’s antigenic sites being bound by maternal anti-D and unable to react in the test. Therefore, Rh typing is unable to be performed and the mother will be given RhIg post-delivery as a precautionary measure.

Whenever a post-partum bleed is suspected, other tests are performed such as the rosette test or Kleihauer-Betke test. The rosette test is a screening test for Rh-Negative mothers and determines whether a bleed greater than 30 mL has occurred. The Kleihauer-Betke test gives a quantitative result for the amount of bleed by microscopically counting the number of fetal cells in a maternal blood sample. A special stain is used for these samples, in which an acid-citrate buffer removes adult hemoglobin from the cells. Any fetal cells present in the maternal blood will appear as bright pink and maternal cells will stain pale pink. For post-partum bleeds in Rh-Negative women, the amount of RhIg can be determined by the percentage of fetal cells in circulation as one RhIg dose would not be sufficient to prevent the production of anti-D.


Kleihauer-Betke test blood smears as viewed under the microscope. It is easy to see the bright pink fetal cells (arrow) within the maternal blood which appear light pink

Overall, I am very thankful for RhIg, as without it, the health of myself and my sisters could have been jeopardized at birth.