Isoimmunization (Rh Disease) in PregnancyWHEC Practice Bulletin and Clinical Management Guidelines for healthcare providers. Educational grant provided by Women's Health and Education Center (WHEC).
When any fetal group factor inherited from the father is not possessed by the mother, antepartum or intrapartum fetal-maternal bleeding may stimulate an immune reaction by the mother. The term hemolytic disease of the fetus/newborn, for instance has replaced hemolytic disease of the newborn because modern diagnostic techniques now allows us to detect the disorder much earlier. Similarly, most experts use the term Rhesus alloimmunization rather than the older expression isoimmunization to describe the formation of maternal antibodies to the Rh D red cell antigen -- reflecting deeper insights into pathophysiology of the disorder. Rhesus (Rh) D blood group incompatibility between the pregnant woman and her fetus is a significant problem due to the possibility of maternal alloimmunization and consequent hemolytic disease of the newborns. The Rh D-negative blood group is found in 15% of whites, 3-5% of black Africans, and is rare in Asians.
The purpose of this document is to understand isoimmunization (Rh Disease) in pregnancy, management and the prevention. To prevent the disease, routine postpartum use of Rhesus immune globulin (Rh I G) in Rh-negative patients was introduced in the United States over 40 years ago. A subsequent recommendation for routine antenatal use at 28 weeks' gestation was introduced 20 years later. Despite these efforts, a recent review of the 2001 birth certificates in the US by the Centers for Disease Control and Prevention indicates that Rh sensitization still affects 6.7 out of every 1,000 live births (1). Maternal immune reactions can also occur from blood product transfusion.
According to the American Medical Association Manual of Style, erythrocyte antigen and phenotype terminology should use single letters or dual letters depending on the antigen in question (eg, O, AB, Le, Rh). A second designation should be used for specific subtypes (eg, Rh D, Rh C). This article uses the designation Rh D to signify the erythrocyte antigen. Women who carry the Rh D antigen are identified as Rh D positive, and those who do not carry the Rh D antigen are identified as Rh D negative. The use of immune globulin to counter Rh D antigen is referred to as anti-D immune globulin (Rh I G). Anti-D isoimmunization remains the most common cause of erythroblastosis fetalis. Antibodies formed in response to the D antigen are of the immunoglobulin G (IgG) type. Consequently, they can cross placenta and hemolyze fetal erythrocytes. Whereas most clinically significant blood group sensitizations noted during pregnancy are still secondary to anti-D incompatibility, sensitization to antigens other than D in the CDE system is not uncommon and can cause severe disease.
Other Clinically Significant Antibodies:
Care of patients with sensitization to antigens other than D that are known to cause hemolytic disease should be the same as that for patients with D isoimmunization. A possible exception is Kell sensitization, in which amniotic fluid analysis has been reported to correlate poorly with the severity of fetal anemia. These patients may benefit from more aggressive fetal assessment; however, optimal management of Kell-sensitized patient is controversial. Lewis (Lea, Leb) and I antigens are not causes of hemolytic disease of the newborn. Isoimmunization resulting from irregular antibodies are (2):
|Blood Group System||Antigen|
|Rh||C, c, e, E|
|Kell||K, k, Ko, Kpa, Kpb, Jsa, Jsb|
|Duffy||Fya, Fyb, Fy3|
|Kidd||jka, jkb, jk3|
|MNSs||M, N, S, s, U, Mia, Mta, Vw, Mur, Hil, Hut|
|P||Yta, Ytb, Lan, Ena, Ge, Jra, Coa, Coa-b-|
|Public antigens||Batty, Becker, Berrens, Biles, Evans, Gonzales, Good, Heibel, Hunt, Jobbins,|
|Private antigens||Radin, Rm, Ven, Wrighta, Wrightb, Zd|
Causes of Rh D Alloimmunization:
Before the introduction of anti-D immune globulin (formerly referred to as Rho[D] immune globulin), hemolytic disease of the fetus and newborn affected 9-10% of pregnancies and was a major cause of perinatal morbidity and mortality. Most women who become alloimmunized do so as a result of fetomaternal hemorrhage of less than 0.1 mL. Several first- and second-trimester clinical events may cause Rh D alloimmunization. Therapeutic and spontaneous abortions are associated respectively with a 4-5% and a 1.5-2% risk of alloimmunization in susceptible (non-alloimmunized) women. Ectopic pregnancy also is associated with alloimmunization in susceptible women. Threatened abortion infrequently causes alloimmunization, clinical procedures, which may breach the integrity of choriodecidual space, also may cause Rh D alloimmunization. Chorionic villus sampling is associated with a 14% risk of fetomaternal hemorrhage, even if the placenta is not traversed. Likewise, cordocentesis and other percutaneous fetal procedures pose a risk for fetomaternal hemorrhage. External cephalic version, whether or not it is successful, results in fetomaternal hemorrhage in 2-6% of cases (3).
Failure to Prevent Rh D Alloimmunization:
In spite of recommendations for immunoprophylaxis, 0.1-0.2% of susceptible Rh D-negative women still becomes alloimmunized. There are two primary reasons for the continuing problem. One reason is failure to implement recommended immunoprophylaxis protocol, resulting in preventable Rh D alloimmunization. The second reason if the small rate (0.1-0.2%) of spontaneous immunization despite the recommended prophylaxis protocol. This problem may become the largest single cause of new Rh D alloimmunization, because alloimmunization from other causes has decreased proportionally (4). Preventable Rh D alloimmunization occurs in susceptible Rh D-negative women for the following three reasons:
- Failure to administer an antenatal dose of anti-D immune globulin at 28-29 weeks of gestation.
- Failure to recognize clinical events that place patients at risk for alloimmunization and failure to administer anti-D immune globulin appropriately.
- Failure to administer or failure to administer timely anti-D immune globulin postnatally to women who have given birth to an Rh D-positive or untyped fetus.
In about 0.1% of deliveries, fetomaternal hemorrhage is in excess of 30 mL; more than the standard dose of Rh I G will be required in these cases. Routine screening of all women for excessive fetomaternal bleeding at the time of delivery is now recommended by American Association of Blood Banks (AABB). Typically this initially involves a sheep rosette test that is read qualitatively as positive or negative. If negative, one vial of Rh I G (300 micro g) is given. If positive, the bleed is quantitated with a Kleihauer-Betke stain or fetal cell stain by flow cystometry. Consultation with the blood bank pathologist to determine the number of doses is encouraged (5). Controversy surrounds the use of Rh I G for threatened abortion. It is probably not indicated when only spotty vaginal bleeding occurs but it should be used in patients with significant clinical bleeding; dose can be repeated in 12-week intervals as necessary. Although a 50 micro g dose can be used up to 13 weeks' gestation, most hospitals no longer stock this preparation and it costs about the same as the standard 300 micro g dose. A second indication of Rh I G that is often overlooked is blunt trauma to the maternal abdomen, particularly at the time of a motor vehicle accident. Finally, if 300 micro g of Rh I G are given late in gestation for external cephalic version or third-trimester amniocentesis for fetal lung maturity, a repeat dose is unnecessary if delivery occurs within 3 weeks, assuming there is no feto-maternal hemorrhage by maternal testing.
Clinical Management of Isoimmunized Patient:
Once it has been established that a pregnant woman is sensitized to an antigen that may cause erythroblastosis, the genotype of the fetus's father should be determined. This is most useful for the atypical antigens because isoimmunization is often secondary to a transfusion. If the father of the fetus does not possess the antigen, the fetus is not at risk. If the father is a heterozygote there is only a 50% chance that the fetus has inherited the blood group antigen and the pregnancy is affected. Maternal serum antibody titers can be measured by a variety of techniques. Agglutination of erythrocytes in saline measures maternal IgM antibody, and this is too large a molecule to cross the placenta. Albumin is a more viscous medium; therefore, the smaller IgG molecules are capable of agglutinating erythrocytes, but the contribution by IgM is not eliminated. The most sensitive and accurate barometer for clinical practice is the indirect Coombs test.
In 1991, once the molecular basis of the Rh D negative blood group became known, prenatal diagnosis of fetal RHD genotype evolved from serologic diagnosis of fetal erythrocytes obtained at cordocentesis to genotypic diagnosis of cells obtained by amniocentesis, a more widely available procedure with a reduced risk of miscarriage. Subsequently, with the demonstration that maternal plasma and serum contain large quantities of cell-free fetal DNA, it became possible to determine fetal RHD genotype non-invasively. This is due to fact that most Rh D negative pregnant women have a deletion of the sequence on both copies of their chromosome 1. Advances in both our understanding of the RHD locus and its variants, as well as technical improvements in the extraction and amplification of cell-free fetal DNA in maternal plasma, have led to incorporation of non-invasive diagnosis of RHD genotype into routine prenatal care in the United Kingdom, France, and the Netherlands (6).
Antibody Titers Measurements:
An antibody titer should be determined at the first prenatal visit, 20 weeks of gestation, and approximately every 4 weeks thereafter. Once the maternal antibody screen returns positive for anti-D, a titer should be ordered. The titer is considered critical if it has been linked to an increased risk of fetal hydrops for a particular institution. An anti-D titer of 1:32 in the first affected pregnancy is often used. However, one should be cautious in interpreting antibody titers; they are only crude estimates of the amount of circulating antibody. When the antibody titer is <1:8, whether directed to D or another paternal antigen capable of causing severe erythroblastosis, no intervention is necessary; when the titer is >1:16 in albumin or 1:32 by indirect antiglobulin (indirect Coombs test), amniocentesis or percutaneous umbilical cord blood sampling (cordocentesis) should be considered. A change of more than one dilution (i.e., 1:4 -- 1:16) represents a true increase in maternal titer. If a patient has had a prior affected pregnancy (neonatal exchange transfusion, early delivery, or intrauterine transfusion), antibody titers are not necessary because amniocentesis or percutaneous umbilical cord blood sampling will be required.
It has become the cornerstone of fetal therapy for hemolytic disease of the fetus and newborn. An early study should be obtained for dating because many of the parameters used to gauge fetal disease -- including ∆OD450 (the change in optical density), peak middle cerebral artery (MCA) Doppler, and fetal hematocrit -- change with gestational age. One of the most significant breakthroughs in recent years has been research that validates the peak systolic MCA Doppler velocity as a reliable screening tool to detect fetal anemia. The vessel can be easily visualized with color flow Doppler. Pulsed Doppler is then used to measure the peak systolic velocity of the MCA just distal to its bifurcation from the internal carotid artery. Enhanced fetal cardiac output and a decrease in blood viscosity contribute to an increased blood flow velocity in fetal anemia. Since the general trend is for the MCA velocity to increase with advancing gestational age, results are reported in multiples of the median (MOMs) much like serum alpha fetoprotein (7).
Amniocentesis and Percutaneous Umbilical Cord Blood Sampling:
Many centers have yet to adopt serial MCA Dopplers. As an alternative, some employ serial amniocenteses for ∆OD450 in conjunction with serial Dopplers until they are comfortable with the latter, as there is a well-established learning curve for the newer procedure. Amniotic fluid bilirubin is most likely derived from fetal tracheal and pulmonary secretions. It can be quantitated by spectrophotometrically measuring absorbance at the 450-nm wavelength in a specimen of amniotic fluid that has been shielded from light. Contamination of amniotic fluid by meconium and by erythrocytes and their porphyrin breakdown products can significantly alter spectrophotometric analysis of 450 nm, but these problems can be largely overcome by chloroform extraction of the amniotic fluid. Heme pigmentation will also generate a peak at the 450 nm wavelength, and in the absence of blood contamination this may be indicative of severe hemolysis. Fetal status is determined by plotting the ∆OD450 measurement on a Liley graph. The recent modification by Queenan and coworkers may be more useful due to its accuracy at gestational ages of less than 27 weeks (8). A rise or plateauing trend into the Rh-positive (affected) zone warrants more invasive testing through cordocentesis. Amniocentesis for lung maturity is widely accepted. Tests for fetal lung maturity such as phosphatidyl-glycerol quantitation, lamellar body count, or the lecithin-sphingomyelin ratio should be employed in cases of rhesus disease as these assays are not affected by excess bilirubin.
Cordocentesis: It was introduced in the mid-1980s; direct access to the umbilical cord vessels by ultrasound guided needle puncture allows clinicians to measure fetal hematocrit, reticulocyte count, bilirubin level, and direct Coombs. Initial enthusiasm for cordocentesis as a primary surveillance tool has waned due to the 1% incidence of fetal loss and a chance for enhanced maternal sensitization. Today, cordocentesis is reserved as a second-line diagnostic tool once amniocentesis or MCA Doppler suggests fetal anemia.
Clinical Management Guidelines:
For a first sensitized pregnancy -- follow maternal titers every 4 weeks up to 24 weeks' gestation; repeat every 2 weeks thereafter. Once a critical value (usually 1:32) is reached, begin serial MCA Dopplers at about 24 weeks' gestation. Alternatively, perform initial amniocenteses every 10 days to 2 weeks for ∆OD450. If the MCA Doppler is >1.5 MOMs or the ∆OD450 value enters the Rh-positive (affected) zone of the Queenan curve, perform cordocentesis with blood readied for intrauterine transfusion for a fetal hematocrit of <30%. Start antenatal testing with non-stress testing or biophysical profiles at 32 weeks' gestation. If repeat MCA velocities remain <1.5 MOMs or ∆OD450 values remain below the Rh-positive (affected) zone, perform amniocentesis at 35 weeks' gestation for ∆OD450 and fetal lung maturity. If mature lungs are found and the ∆OD450 value has not reached the Rh-positive (affected) zone; induce at 37 weeks' gestation to allow for hepatic maturity in an effort to prevent hyperbilirubinemia. If immature lungs are found and ∆OD450 has reached the Rh-positive (affected) zone, treatment with 30 mg of oral phenobarbital three times a day and labor can be induced in one week. This will accelerate fetal hepatic maturity and allow for more efficient neonatal conjugation of bilirubin (9). In these cases, a unit of packed RBC cross-matched to the pregnant patient should be prepared prior to delivery so that it is available if the pediatrician must do an emergency neonatal transfusion. If immature lungs are found and ∆OD450 is not in the Rh-positive (affected) zone, repeat the amniocentesis at 37 weeks.
For a previously affected fetus of infant that has had a transfusion -- maternal titers are not helpful in predicting the onset of fetal anemia after the first affected gestation. In cases of a heterozygous paternal phenotype, perform amniocentesis at 15 weeks' gestation to determine the fetal Rh D status. If an Rh D-negative fetus is found and paternity is certain, no further testing is needed. Begin with MCA Doppler assessment or serial amniocenteses for ∆OD450 at 18 weeks' gestation. Repeat at 1 to 2 week intervals. If a rising value of MCA Doppler >1.5 MOMs or rising ∆OD450 value into the Rh-positive (affected) zone is noted, perform cordocentesis with blood readied for intrauterine transfusion for fetal hematocrit of <30%.
Intrauterine Fetal Transfusion:
Initially, clinicians used the peritoneal cavity to do these transfusions; in non-hydropic fetus, the rate of absorption was estimated to be 10-15% per 24 hours. Absorption was slower if hydrops was already evident. Because of erratic absorption, especially in hydropic fetuses intravascular fetal transfusion has largely replaced the intraperitoneal technique. As experience with cordocentesis accumulated, the direct intravascular transfusion (IVT) of donor red cells into the fetal umbilical vein at its placental insertion became the most common method of intrauterine transfusion in the United States. Some US centers combine intravascular transfusion with the original intraperitoneal method in an effort to prolong the intervals between procedures. Typically, a fresh type O, Rh D-negative unit is screened to be sure it does not contain cytomegalovirus antibodies and packed to a hematocrit of 75% to 85%. This step allows a minimal blood volume to be administered to the fetus during transfusion. The blood is then leuko-reduced with a special filter and irradiated with 25 Gy to prevent graft-vs-host reaction. Data on the neuro-development of neonates transfused by intravascular transfusion are limited. Most studies point to more than a 90% chance of intact survival (10). Hydrops fetalis does not seem to affect this outcome. Sensineural hearing loss may be slightly increased due to prolonged exposure of the fetus to high levels of bilirubin. A hearing screen should be performed during the early neonatal course and repeated by two years of life.
Encouraging is the improved outcome for hydropic fetuses, although these results are promising, there are complications with any invasive procedure. Even in the most experienced hands, percutaneous umbilical cord blood sampling alone results in procedure-related pregnancy loss of approximately 1%. The procedure-related mortality for intravascular transfusion has been reported to be between 4% and 9%. Additional significant morbidity has included prolonged fetal heart rate decelerations that required emergency cesarean section and increases in maternal antibody titer, presumably secondary to fetal-maternal hemorrhage. When weighing the procedure-related risks against those for the neonate in the nursery, one should seriously consider delivery rather than performing an intravascular transfusion after 34 completed weeks of pregnancy.
Cost-Effectiveness of Rh D Prophylaxis Programs:
Economic analysis of anti-D immune globulin prophylaxis is based on the cost of anti-D immune globulin and the number of alloimmunizations that would be prevented. In summary, the cost-effectiveness of antenatal Rh D immune globulin to all Rh D-negative pregnant women and in all circumstances wherein fetomaternal hemorrhage might occur has not been proven. Available data support that third-trimester antenatal prophylaxis is cost-effective in primigravidas. As long as the supply of anti-D immune globulin is adequate and data do not exist to support other recommendations, most experts believe that it is unethical to withhold anti-D immune globulin from any patient at risk of Rh D alloimmunization (11). The risk of excessive fetomaternal hemorrhage exceeding 30 mL (the amount covered by the standard 300 micro g dose of anti-D immune globulin) at the time of delivery is approximately 1 in 1,250. Pregnancies designated as high risk should be screened for excessive fetomaternal hemorrhage, including cases of abdominal trauma, abruptio placentae, placenta previa, intrauterine manipulation, multiple gestation, or manual removal of placenta. However, such a screening program has been reported to detect only 50% of patients who required additional anti-D immune globulin. Based on this finding, the American Association of Blood Banks has recommended that all Rh D-negative women who deliver Rh D-positive infants be screened using the Kleihauer-Betke or rosette test (12).
Autism risk with anti-D Rh immunoglobulin exposure:
Until 2001, anti-D IgG contained thimerosal in a concentration of 0.003% (10.5 micrograms of ethyl mercury, on average). This compound has been present in a number of vaccines given to infants and young children and has been studied as a possible causal factor in the development of autism spectrum disorders. To date, most studies have eliminated this possibility and the findings of the current study appear to concur with previous findings (13). The authors conducted a case-control study examining prenatal, perinatal, and neonatal risk factors for autism spectrum disorders. They identified 420 children with at least one diagnosis of autism spectrum disorders born between 1995 and 1999 who were monitored for at least 2 years after birth. Each child was matched with five controls without autism spectrum disorders for sex, year of birth, and hospital. The authors gathered information on maternal Rh status and anti-D immunoglobulin G (IgG) exposure during pregnancy and determined whether the mother had received and influenza vaccination during pregnancy. The proportion of Rh-negative mothers did not differ between patients and controls (11.5% versus 10%) and single and multiple variable adjustments for sex, maternal age, maternal race or ethnicity, maternal education, parity, and plurality did not alter the results. No association between maternal Rh status and risk of autism spectrum disorders was observed for subgroups of children defined by sex, plurality, autism severity, birth order, or number of affected children within siblings. The number of total anti-D IgG injections received was similar for affected mothers and control mothers.
The authors concluded that there was no association between maternal Rh status, prenatal anti-D IgG exposure, and autism spectrum disorders. The authors attempted to find a second source of mercury in these children by determining if the mother had received an influenza vaccine containing thimerosal as a preservative, but they did not investigate other sources of mercury exposure, such as fish consumed by mothers or dental amalgam in teeth fillings. Nevertheless, without being able to correct for these factors, the prenatal use of anti-D IgG did not appear to influence the development of autism spectrum disorders in children (14).
The Rh D-negative woman who is not Rh D-alloimmunized should receive anti-D immune globulin: at approximately 28 weeks of gestation, unless the father of the baby is also known to be Rh D negative; within 72 hours after the delivery of an Rh D-positive infant; after a first-trimester pregnancy loss; and after invasive procedures such as chorionic villus sampling, amniocentesis, or fetal blood sampling. Anti-D immune globulin prophylaxis should be considered if the patient has experienced threatened abortion; second- or third- trimester antenatal bleeding; external cephalic version or abdominal trauma. The reduction in the incidence of Rh D alloimmunization is a prototype for the effectiveness of preventive medicine.
Acknowledgement -- gratitude is expressed to Dr. Maria M. Morales, Professor Titular de Universidad, University of Valencia, Spain, Collaborator with Spanish Ministry of Health, for her expertise, support and friendship in preparation of this chapter.
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- Serrano MA, Bayon JE, Pascolo L et al. Evidence for carrier-mediated transport of unconjugated bilirubin across plasma membrane vesicles from human plancental trophoblast. Placenta 2002;23:527-535
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- Breecher ME. Technical Manual of American Association of Blood Banks. Bethesda, MD: American Association of Blood Banks, 2002
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- Mari G, Deter RL, Carpenter RL et al. Noninvasive diagnosis by Doppler ultrasonography of fetal anemia due to maternal red-cell alloimmunization. Collaborative Group for Doppler Assessment of the Blood Velocity in Anemic Fetuses. N Engl J Med 2000;342:9-14
- Sikkel E, Vandenbussche FP, Oepkes D et al. Amniotic fluid Delta OD 450 values accurately predict severe fetal anemia in D-alloimmunization. Obstet Gynecol 2002;100:51-57
- Trevett T, Dorman K, Lamvu G et al. Does antenatal maternal administration of phenobarbital prevent exchange transfusion in neonates with alloimmune hemolytic disease? Am J Obstet Gynecol 2003;189:S214
- van Kamp IL, Klumper FJ, Meerman RH et al. Treatment of fetal anemia due to red-cell alloimmunization with intrauterine transfusions in the Netherlands, 1998-1999. Acta Obstet Gynecol Scand 2004;83:731-737
- ACOG Practice Bulletin. Management of alloimmunization during pregnancy. Number 75, August 2006
- Moise KJ. Management of rhesus alloimmunization in pregnancy. Obstet Gynecol 2008;112:164-176
- Croen LA, Matevia M, Yoshida CK et al. Maternal Rh D status, anti-D immune globulin exposure during pregnancy, and risk of autism spectrum disorders. Am J Obstet Gynecol 2008;199:234.e1-234.e6. (Level II)
- ACOG Clinical Review. Autism risk with anti-D Rh immunoglobulin exposure. Volume 14; Issue 2 March-April 2009
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