Hemolytic disease of the newborn (HDN)

The Hemolytic disease of the newborn (HDN) is an autoimmune condition in which the fetus's red blood cells have been targeted and lysed by the mother’s antibodies. This leads to neonatal jaundice, which is characterized by the yellow color in the skin, eyes and even mucus membranes. The yellow pigments are due to increased levels of blood bilirubin, which is normally a yellow breakdown product of old red blood cells. HDN was first described in1609 by a French midwife, in which a newborn died. However, the mechanism behind HDN was not discovered until 1950s. 
The picture from innermostsecrets.com demonstrates the known HDN mechanism.

Immunology of HDN

The Rh system is an important blood-group system. The most significant Rh antigen on the surface of the red blood cells is the D antigen (RhD), which makes a blood group to be positive (e.g. O+, A+, B+ or AB+).  The HDN can happen when a RhD-negative mother becomes pregnant with an RhD-positive child. During pregnancy, the mother's immune system can generate antibodies against the fetal RhD antigen. These antibodies can transport across placenta and thus enter the fetal circulation. Basically, the transferring of mother’s antibodies to the fetus via placenta and then milk is an important mechanism for protecting fetus and newborns, which do not have a mature and strong immune system. However, this may also lead to fetal red blood cell lysis or HDN if there is an incompatibility between the mother and baby.

The mother’s immune system should first be sensitized to RhD antigens in order to generate antibodies against these antigens. An RhD-negative mother can be sensitized during the pregnancy or after receiving RhD-positive blood by a blood transfusion. After sensitization the mother’s blood will contain anti-RhD. Entering a small amount of fetal blood in the mother's circulation is enough to sensitize the mother. This normally happens during the delivery of the RhD-positive child. Sensitization can also occur during the pregnancy by prenatal bleeding or a miscarriage, medical procedures etc. The risk of sensitization to the RhD antigen is decreased if the fetus is ABO incompatible because fetal cells can be more rapidly targeted by the mother’s natural anti-A and/or anti-B antibodies, reducing the risk of recognition of the fetal RhD antigen by the mother’s immune system. 

The first maternal anti-RhD antibody is of the IgM type, which can not pass the placenta. Thus the first RhD-positive baby is not normally affected. However, in subsequent pregnancies, the RhD antigen can stimulate the production of anti-RhD antibody of the IgG type, which can pass across the placenta and enter the fetal circulation. 

In rare cases, an incompatibility of the ABO blood group may cause HDN. This can be the case especially when a mother with blood type O becomes pregnant with a fetus with a blood type A, B, or AB. In these cases, the mother's natural anti-A and anti-B antibodies can cross the placenta and lyse the fetal red blood cells.

Diagnosis

The Coombs tests confirm the presence of anti-RhD antibodies in the mother’s blood and thus show the prior sensitization of the mother to RhD antigens. The neonatal or cord blood gives a positive direct Coombs test and the maternal blood (serum) gives a positive indirect Coombs test. The test is called Coombs because Robin Coombs developed the method of using antibodies that are targeted against other antibodies.

The direct Coombs test or the direct anti-globulin test (DAT) is used to show if antibodies have bound to the red blood cells' surface antigens. In this test, the newborn’s red blood cells should be incubated with anti-human antibodies (Coombs reagent). If this produces cause agglutination of the cells, the direct Coombs test is positive demonstrating that the newborns red blood cells were already covered by the mother’s antibodies. This is called the direct Coombs test because the anti-Immunoglobulin binds "directly" to the maternal anti-RhD antibodies, which have already coated the fetal red blood cells.

The indirect Coombs test also known as indirect anti-globulin test (IAT) is used in testing of pregnant women. It detects anti-RhD antibodies in the mother’s blood. In this case, the mother's serum should be incubated with the RhD-positive red blood cells (type O) and then washed red blood cells should be incubated with anti-human antibodies (Coombs reagent). If agglutination occurs, the indirect Coombs test is positive because it shows that the mother’s serum contained anti-RhD antibodies. This is called the indirect Coombs test because this is an indirect evidence for the presence and ability of maternal anti-RhD antibodies to bind to fetal red blood cells. 

The picture from immunology.cam.ac.uk demonstrates the coombs tests' procedures.

Treatment

The therapeutic antibodies, which can remove the mother's anti-RhD antibodies from the mother's circulation can be a possible treatment method. However, treatment of pregnant women with anti-RhD antibodies that bind and destroy the fetal RhD positive red blood cells that have entered the maternal circulation can largely prevent the development of HDN. Usually, mothers are injected with anti-RhD antibody at about 28 weeks gestation, when fetal red blood cells express the D antigen. The second injection occurs at about 34 weeks. The last dose of anti-RhD is given after delivery. Blood transfusions may also be required to solve the problems with anemia and high bilirubin levels in newborns.