Rh Sensitivity During Pregnancy Ziqi Liu, Jungin Park, Sharon Saini, Christina Truong October 9th, 2018 PHM142 Fall 2018 Instructor: Ms. Maya Latif Coordinator: Dr. J. Henderson

Rh blood group Two genes involved: RHD and RHCE 49 different Rh antigens, the most common include: D, C, E,c and e Antigens are present on the surface of RBCs using a tetrameric protein complex involving RHAG and Rh proteins (RhD/ RhCE) Rh antigens maintain integrity of red blood cells Similar to the ABO blood group, there is also the Rh blood group that is important in determining a person’s blood type. There are 2 main genes involved in the RH blood group including RHD and RCE. Both these genes are highly polymorphic and due to many genetic rearrangements there is a lot of variety in the Rh antigens. Up to now there are 49 antigens that are part of the Rh blood group, with the most common one’s being D, C, E, c, and e. These antigens are present on the surface of red blood cells through a tetrameric protein complex: which involves RHAG (Rh associated glycoprotein) and 2 Rh proteins (RhD or RhCE). Rh antigens help maintain the integrity of RBCs, and rbcs without these antigens have an abnormal shape (increased osmotic fragility, shortened life span)

Rhesus (Rh) factor The RHD gene on chromosome 1 encodes for RhD protein which carries the D antigen Rh factor is inherited and passed from parent to child Individual with the D antigen= Rh positive Individual without the D antigen= Rh negative For this presentation we will be focusing on the D antigen which is also referred to as the Rh factor. There is the RHD gene present on chromosome one that is responsible for encoding RHD which is the protein that carries the D antigen. The RHD gene has a dominant mode of inheritance and it is passed on from parent to child. Individuals with the D antigen are said to be Rh positive, and individuals without the D antigen are Rh negative.

Demographics Rh Negative Frequencies: 15-17% Caucasians, 5% Africans, 3% Asians Caucasians → removal of the RHD gene Africans & Asians → inactivation or silencing of the RHD gene The frequency of the D antigen varies among different populations. For example the frequency of being Rh negative in Caucasians, Africans , and Asian populations is 15-17%, 5%, and 3% respectively. This then means that being Rh negative is prevalent in higher frequencies in Caucasians. For Caucasian individuals who are Rh negative, it is due to the removal of the RHD gene from the chromosome therefore it is not present. However, in African and Asian populations, individuals who are Rh negative, it caused by the silencing or the inactivation of the RHD gene can be caused by premature stop codon or inheriting a hybrid RHD gene There are also very rare phenotypes known as Weak D and partial D with individuals who have lower expression of the RHD gene

Antibodies Antibodies are produced by the plasma cells in the body against antigens Rh antigens are immunogenic Rh- individuals produce anti-D antibodies when they encounter the D antigen The most common antibodies produced against the Rh antigen are IgG When anti-D antibodies come into contact with the D antigen, they mark the RBC for destruction by the immune system As discussed previously in lecture, antibodies are produced by plasma cells in the body in response to foreign antigens. Rh antigens are said to be very immunogenic, which means they are known to highly provoke the immune system to induce a response - Individuals that are Rh negative will produce anti-D antibodies when they encounter the D antigen. - The most common antibodies that are produced against the Rh antigen are IgG (and IgM), these antibodies are not present in the blood of a Rh negative individual when they have not been exposed to the D antigen.

Rh Sensitization Rh sensitization occurs when an Rh- mother has Rh+ child and is exposed to the fetal blood. Primary mechanism of exposure is during delivery, but can occur during ectopic pregnancy, ruptures in placenta, transfusions, etc. She develops anti-D antibodies during and after this first pregnancy. Rh sensitization can only occur when the mother is Rh negative and the baby is Rh positive. If the father is heterozygous, there is a 50% chance that the child will be Rh positive. However, if the father is homozygous dominant, there is a 100% chance the child will be Rh positive. The most common way for fetal blood to get into maternal circulation is during delivery. The placenta is usually a good barrier but after delivery, the placenta gets damaged and there is some leakage of fetal blood into the maternal circulation. As a result, the mother develops anti-D antibodies because of this exposure and becomes Rh sensitized.

Pathogenesis of Rh Sensitization After sensitization, the mother has anti-D antibodies in her immune repertoire. If she gets pregnant again with another Rh+ child, she can readily produce anti-D antibodies when her immune system recognizes the D antigen on the child’s RBCs. The mother’s immune system will now attack fetal RBCs. Rh sensitization does not typically cause harm during the first pregnancy, but it is basis of the complications in future pregnancies if the second child is also Rh+. After sensitization, the mother now has memory for anti-D antibodies in her immune system. If she gets pregnant again with another Rh+ child, the anti-D antibodies she produced will recognize the D antigen on the child’s RBC and will proceed to attack them.

Mechanism of IgG Placental Transcytosis IgG endocytosis into the apical membrane of the placenta. IgG binds FcRN receptor inside the endosome. Endosome fuses with basal membrane and releases IgG into fetal circulation. IgG binds to Rh+ fetal RBC and leads to hemolysis. This process is dependent on the change in pH. Anti-D IgG is taken up by endocytosis on the apical membrane of syncytiotrophoblast; which is the outer layer of the placenta. This forms an endosome which has an acidic pH of 6.0 and allows the binding of the Fc portion of IgG with a neonatal FcRN receptor. IgG is then transcytosed to the basal membrane where it returns to normal physiological pH of 7.4. IgG then is released from its receptor and into fetal circulation. As a result, IgG binds with Rh+ fetal RBCs and forms antigen-antibody complexes and leads to alloimmune hemolytic anemia.

Consequences Can range from mild to severe Mild: anemia, reticulocytosis Severe: hemolytic disease of the newborn (HDN), hydrops fetalis or stillbirth Importance of blood tests at initial prenatal screening to determine whether mother is Rh- and/or Rh-sensitized Some countries lack prophylactic programs for Rh sensitization during pregnancy, thus Rh HDN is still prevalent.

Prevention Rho(D) Immune Globulin Injection (RhIG) Solution of IgG anti-D antibodies Anti-D antibodies bind and inactivate fetal Rh antigens before they stimulate immune response in the mother Prevents maternal B-lymphocyte activation and memory cell formation Given both during and following pregnancy (around 28th week of pregnancy and also within 72 hours of baby’s birth) Only works for a short time, so injection is needed each time you get pregnant Ex) RhoGAM, WinRho

Treatment for Affected Newborns Phototherapy Lowers bilirubin levels in the baby’s blood by a process called photo-oxidation Results in the baby’s liver to break down and remove the bilirubin from their blood easily Exchange transfusion The baby’s blood is replaced with blood from a suitable matching donor Removes bilirubin and hemolytic antibodies Intravenous immunoglobulin (IVIG) Pooled antibodies from multiple donors Deplete maternal antibodies Prevents RBCs from destruction, so the level of bilirubin will stop rising. Phototherapy: UV light exposure used to treat newborn jaundice. Lowers bilirubin levels in the baby’s blood by a process called photo-oxidation. Photo-oxidation adds oxygen to the bilirubin. Results in the baby’s liver to break down and remove the bilirubin from their blood easily Exchange transfusion: Used when the baby has a very high level of bilirubin or as a secondary treatment option if phototherapy hasn’t been effective. The baby’s blood is replaced with blood from a suitable matching donor. It removes bilirubin and hemolytic antibodies and corrects anemia. Intravenous immunoglobulin (IVIG): Pooled antibodies from multiple donors, deplete maternal antibodies. Only used if phototherapy alone hasn’t worked and the level of bilirubin in the blood continues to rise. Prevents red blood cells from destruction, so the level of bilirubin will stop rising.

Summary 2 genes of the Rh blood group include RHD, and RHCE → The RHD gene encodes for the RhD protein that carries the D antigen. The D antigen is passed from parent to child. Individuals without the D antigen= Rh negative. Individuals with D antigen= Rh positive. Rh sensitization occurs when the mother is Rh- and the fetus is Rh+ and is exposed to blood from the fetus. The mother develops anti-D antibodies which attack the fetus RBC in subsequent Rh+ pregnancies. Maternal anti-D IgG crosses the placental membrane into fetal circulation through a pH dependent process. Consequences can be severe and lead to stillborn babies. Prevention: Rho(D) immune globulin injection (RhIG) Treatments: Phototherapy, exchange transfusion, intravenous immunoglobulin

References Antonios, Nathalie, "Rh Incompatibility in Pregnancy". Embryo Project Encyclopedia (2011-04-08). ISSN: 1940-5030 http://embryo.asu.edu/handle/10776/2073. Ascari, William Q. “Rho (D) Immune Globulin (Human).” Jama, vol. 205, no. 1, Jan. 1968, p. 1., doi:10.1001/jama.1968.03140270025004. Barclay, Sheilagh. “Red Blood Cell Antigens and Human Blood Groups.” Handbook of Transfusion Medicine, 2001, pp. 91–106., doi:10.1016/b978-012348775-9/50062-4. Costumbrado, John, Ghassemzadeh, Sassan. “Rh Incompatibility.” StatPearls. Treasure Island (FL): StatPearls Publishing. 2018. Available from: https://www.ncbi.nlm.nih.gov/books/NBK459353/ “Stanford Children's Health.” Stanford Children's Health - Lucile Packard Children's Hospital Stanford, www.stanfordchildrens.org/en/topic/default?id=hemolytic-disease-of-the-newborn-90-P02368. Tanyer, G. “Brief Report. Multiple Dose IVIG Treatment in Neonatal Immune Hemolytic Jaundice.” Journal of Tropical Pediatrics, vol. 47, no. 1, Jan. 2001, pp. 50–53., doi:10.1093/tropej/47.1.50. “Using Rh Immune Globulin in Pregnancy to Prevent Rh Disease.” Atlas of Science, atlasofscience.org/using-rh-immune-globulin-in-pregnancy-to-prevent-rh-disease/. Westhoff, Connie M. “The Structure and Function of the Rh Antigen Complex.” Seminars in Hematology, vol. 44, no. 1, 2007, pp. 42–50., doi:10.1053/j.seminhematol.2006.09.010. Wilcox, Christopher R., Beth Holder, and Christine E. Jones. “Factors Affecting the FcRn-Mediated Transplacental Transfer of Antibodies and Implications for Vaccination in Pregnancy.” Frontiers in Immunology 8 (2017): 1294. PMC.

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