1. Rh ISOIMMUNIZATION By: DR
Rh ISOIMMUNIZATION By: DR. MALAK AL-HAKEEM Associate Professor & consultant obstetrics & gynecology.

2. What is isoimmunization? Why is it a concern during pregnancy?
Isoimmunization refers to the maternal development of antibodies to fetal red blood cell (RBC) antigens. During normal pregnancy a small number – and in some situations a large number – of fetal RBCs cross the placenta and enter the maternal circulation. When the fetal RBC antigens differ from those of the mother, a maternal immune response is generated. Although this response may be relatively small at first exposures, elicit a larger and quicker response of IgG. This maternal IgG, directed at the fetal RBC antigen, is capable of traversing the placenta into the fetal circulation, causing hymolysis.

3. What is hemolytic disease of the newborn
What is hemolytic disease of the newborn? Most often secondary to isoimmunization to the Rh (rhesus) antigen, fetal RBCs, that contain an antigen foreign to the mother (such as an RhD-positive fetus with an RhD – negative mother) become a site of binding for the maternal IgG. Once the maternal IgG binds to the RhD antigen on the fetal RBCs, these cells are destroyed. Depending on the severity of this hemolysis, the fetus may become anemic and hydropic, ultimately resulting in stillbirth.

4. Why does the severely anemic fetus become hydropic?
Severe fetal anemia results in extramedullary production of RBCs (primarily in the fetal liver and spleen). Extensive hepatic erythropoiesis distorts the portal venous circulation, leading to obstruction and finally to hepatomegaly, ascites, and placental edema. Hypoalbuminemia secondary to liver dysfunction also may contribute to this generalized edema.

5. In addition to anemia, what are the risks to the RhD-positive newborn whose mother is sensitized? Marked hyperbilirubinemia, which, if untreated, may lead to central nervous system damage.

6. How many red blood cell (RBC) antigens are there?
More often 400 antigens have been identified on the red blood cell surface. However, only a few are clinically important as causes of hemolytic disease of the newborn. In addition to the Rh antigen, others include A, B, Kell (k), Duffy (Fy), Kidd (Jk), M,N,S, Lutheran (Lu), Diego (Di), and Xg.

7. What is the most common cause of hemolytic disease of the newborn?
Rh antigen isoimmunization

8. How many antigens comprise the Rh system on the human red blood cell?
Five: CDE and c,e. Small d has not been identified. There are three closely linked loci within the Rh complex (D,C,E) with allelic forms (d,c,e). Some consider each antigen to be a separate gene. These three sites tend to be inherited intact with little crossover and rearrangement

9. In which population is RhD – negative trait most common
In which population is RhD – negative trait most common? In which population is it rarely encountered
Basque people are 95% RhD – negative, whereas approximately 15% of Caucasians are RhD – negative. Less than 5% of Asians and North American Indians are RhD – negative.

10. Define “D”
D” refers to the situation in which “D” is not detected by anti-D sera at a level that types the person as RhD – positive. This situation can occur by two mechanisms. First, the person may genetically contain the D sequence, but its expression is decreased (or masked) by the presence of C at the rhesus loci on the other chromosome. These genetic RhD – positive individuals are not at risk for isoimmunization. Less often, however, “D” is the result of genetic absence of a portion of the D antigen. Such women are at low risk for isoimmunization. RhoGAM should be given to the “D” mother unless the “environmental” (C allele) can be differentiated from the “genetic” variety.

11. What is the difference between a direct and indirect Coombs’ test in the fetus?
A direct Coombs’ test detects the presence of attached immunoglobulin in red blood cells; an indirect Coombs test detects the presence of free immunoglobin in sera.

12. What is immunoglobulin prophylaxis for RhD-negative, nonsensitized mothers? Why is it helpful?
It is RhD immune globulin (RhoGAM) prepared from subjects previously sensitized to RhD. It absorbs the fetal RhD-positive antigen thus blocking formation of maternal Rh antibody at the time of exposure, usually at delivery. An alternative theory is that RhoGAM works at the cellular level of IgG production.

13. The standard dose (300 µg) of RhoGAM given after delivery provides coverage for how large of a fetomaternal hemorrhage?
30 ml of fetal whole blood or 15 ml of fetal packed red blood cells.

14. What percentage of women have evidence of fetomaternal hemorrhage after delivery? In what percentage is this bleeding considered excessive (> 5ml of fetal blood)?
Seventy – five percent have evidence of fetomaternal hemorrhage. It is > 5 ml in < 1%.

15. In what circumstances may a woman at delivery have experienced fetomaternal hemorrhage of > 15 ml ( 30 ml of fetal whole blood)?
- Placental abruption
- Placenta previa associated with bleeding
- Manual removal of the placenta
- Multifetal gestations

16. Which test can be used to estimate the amount of fetomaternal hemorrhage?
Kleihauer – Betke test. A maternal blood sample can be treated with an acid dilution procedure, allowing identification and quantification of fetal red blood cells.

17. After a first pregnancy in a woman who is RhD – negative with an RhD – positive fetus and who does not receive RhoGAM, what is the risk that she will become sensitized?
Approximately 18%

18. What is sensibilization?
Approximately one-half of women who are RhD-negative, do not receive RhoGAM after their first RhD – positive pregnancy, and become sensitized to the RhD antigen respond immediately with immunoglobin production to the RhD antigen. The other half mount a small, often undetectable response and may quickly mount an immune response with subsequent exposure to the antigen.

19. After evacuation of a molar gestation, do women who are RhD – negative need RhoGAM?
With complete molar gestation, there is theoretically no production of fetal red blood cell, the only cells that expresses Rh antigen. However, the distinction of a complete mole with no fetal red blood cells in often not available at the time the decision for RhoGAM is needed. For this reason, many advocate RhoGAM even after evacuation of a complete mole.

20. How should pregnant women be followed to avoid fetal complications from Rh antibodies?
At their first prenatal visit, all pregnant women should be typed according to blood group, with serum screening for antibodies (indirect Coombs) to all RBC antigens associated with hemolytic disease of the newborn. For RhD-negative mothers without antibodies, antibody status should reevaluated at least once during pregnancy, generally at weeks. Some recommend reevaluation of antibody status each trimester. RhoGAM is administered to RhD-negative women at weeks to prevent the small risk of sensitization in the third trimester.

21. For RhD – negative women with evidence of antibodies, serial antibody titers are obtained on a monthly basis. Further evaluation of the fetus is initiated when the antibody titers reach a critical level (≥ 1:16 in most laboratories). If there is a history of a previously affected sibling in utero, many initiate further testing of the fetus at 4-6 weeks before the gestational age at which the sibling was affected.

22. How is amniocentesis used in the management of Rh isoimmunization?
Amniocentesis can serve two purposes:
1. Assessment of fetal Rh status by DNA-based molecular studies of the amniocytes and
2. Measurement of bilirubin pigments in the amniotic fluid.

23. Amniotic bilirubin reflects ongoing erythrocyte destruction
Amniotic bilirubin reflects ongoing erythrocyte destruction. It can be quantitated by measuring the absorbance of amniotic fluid on a continuously recording spectrophotometer at the 450-nm wave length (∆ OD450). The ∆OD450 is determined by measuring the absorbance of amniotic fluid at 365nm and 535nm and drawing a straight line. The ∆OD450 is then determined by subtracting the actual value from the expected 450nm value. Liley defined three zones that relate the fetal health to the bilirubin level and the suggested course of action for each zone. For the premature fetus with evidence of significant hemolysis based on amniotic fluid bilirubin analysis, assessment of the fetal hematocrit by percutaneous umbilical blood sample (PUBS) is the next step.

24. What is the risk that after amniocentesis a RHD – negative mother will become sensitized if she does not receive RhoGAM?
Even with placental localization by ultrasound and avoidance of the transplacental passage of the needle, amniocentesis is associated with a 1-2% risk of fetomaternal hemorrhage of ≥ 1.0 ml fetal RBC. This risk is similar for amniocentesis performed in the second trimester for genetic indications and in the third trimester. Given these risks, RhoGAM should be administered to unsensitized Rh-negative women after amniocentesis. Of note, in the previously Rh-sensitized woman, even this small degree of fetomaternal hemorrhage can be associated with a marked increase in antibody titers.

25. What factors are considered in transfusing an anemic fetus?
In general, a fetal hematocrit below 25% warrants transfusion if gestational age is remote from term. To determine the amount of RBCs to transfuse, the gestational age and weight of the fetus, total vascular volume of both fetus and placenta, and desired final hematocrit are incorporated into the calculation. For transfusion, O-negative, CMV-negative, irradiated blood is used.

26. How are in utero fetal transfusion performed?
Either intravascularly or intraperitoneally. The technique is essentially similar, with ultrasound visualization of placement of a 20-gauge spinal needle within the amniotic sac and further advancement of the needle into either the umbilical vein (intravascular transfusion) or the peritoneal cavity of the fetus (intraperitoneal).

27. What are the advantages and disadvantages of intravascular transfusion?
The advantages of intravascular transfusion are the ability to obtain a fetal hematocrit before and after procedure and direct placement of the transfused packed red blood cells into the fetal vascular system with more rapid correction of the fetal hematocrit. Disadvantages include technical constraints due either to vessel size at early gestational age or posterior placentation and dislodgment of the needle from the umbilical vein by movement during the procedure.

28. Can a fetus still be transfused if it is hydropic?
Yes. If an intraperitoneal transfusion is needed, the ascitic fluid may be removed before transfusion.

29. What are the risks of fetal transfusion and the anticipated complications?
The risk of procedure – related mortality is 4-9%. In addition, significant morbidity may include fetal bradycardia, abruption, preterm rupture of membranes, and emergent delivery due to fetal distress.

30. When are fetuses who have required intrauterine transfusion delivered?
Once fetal lung maturity has been obtained, the risk of intrauterine transfusion outweighs the risk of delivering the infant with transfusion performed in the neonatal intensive care unit.

31. Why do the lungs in infants with Rh sensitization generally mature at a slightly later gestational age?
The cause is unknown but may be related to hydropic changes in the placenta with increased insulin production, an etiology similar to delayed fetal lung maturation in insulin – dependent diabetics

32. Can the Rh type of a fetus be determined early in pregnancy?
For several of the Rh antigens it is now possible to determine the Rh antigens status of a fetus through DNA molecular probes with chorionic villus sampling or amniocentesis. This technique allows the opportunity to determine RhD-antigen status of a pregnancy early in gestation and may be considered by couples who have had prior pregnancies complicated by intrauterine transfusion or fetal demise. The expectation is that subsequent pregnancies would be similarly or more severely affected.

33. Because RhD immunoglobulin (RhoGAM) has prevented much of the isoimmunization problem, what remains?
The Kell antigen, Kell type is not determined before blood transfusion. Approximately 10% of people are Kell – positive, suggesting that the risk of sensitization to Kell after a blood transfusion is approximately 10%.

34. How does ABO sensitization differ from RhD sensitization?
Although successive pregnancies with fathers homozygous for RhD usually become more severe, ABO sensitizations are the opposite: the first pregnancy is usually the worst.

35. What are the private antigens?
In rare instances, a fetus may have inherited a rare red blood cell surface antigen from the father. As the red blood cells of the mother in all likelihood do not contain this antigen, during her first pregnancy she may become sensitized to this “private” antigen. Subsequent pregnancies, depending on the antigen’s ability to elicit an antibody response, are at variable risk for isoimmunization.

36. END OF LECTURE GOOD LUCK!