Clinical Expert Series

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Management of Rhesus Alloimmunization in Pregnancy

Kenneth J. Moise Jr,
MD

Rhesus immune globulin has decreased the prevalence of rhesus D alloimmunization in pregnancy so that only approximately six cases occur in every 1,000 live births. The rarity of this condition warrants consideration of consultation with or referral to a maternalfetal medicine specialist with experience in the monitoring and treatment of patients with red cell alloimmunization in pregnancy. Evaluation for the presence of maternal anti-D antibody should be undertaken at the first prenatal visit. First-time sensitized pregnancies are followed with serial maternal titers and, when necessary, serial Doppler assessment of the peak systolic velocity in the middle cerebral artery. In cases of a heterozygous paternal genotype, new DNA techniques now make it possible to diagnose the fetal blood type through free fetal DNA in maternal plasma. When there is a history of an affected fetus or infant, maternal titers are no longer predictive of risk in subsequent pregnancies. Serial peak middle cerebral artery velocities using Doppler ultrasonography can be used in these pregnancies to detect fetal anemia. In some situations, intrauterine transfusion is necessary through ultrasound-directed puncture of the umbilical cord with the direct intravascular injection of red cells. Perinatal survival rates of more than 90% have been reported; hydrops fetalis reduces the chance for a viable outcome by up to 11%. Neonatal and infant outcomes are complicated by the need for repeated transfusions secondary to suppressed erythropoiesis. Long-term studies have revealed normal neurologic outcomes in more than 90% of cases. Future therapy will involve selective modulation of the maternal immune system, making the need for intrauterine transfusions a rarity.
(Obstet Gynecol 2008;112:16476)

hesus alloimmunization in pregnancy has become a rare encounter in clinical practice. Once a major contributor to perinatal morbidity and mortality, the widespread adoption of guidelines for the antenatal and postpartum use of rhesus immune globulin has had a major effect on this disease. Advancing ultra-

sonography and genetic technologies have revolutionized the contemporary management of the sensitized patient. Management of the pregnant patient found to have anti-red cell antibodies other than anti-D is beyond the scope of the current review.

Prevalence
The exact prevalence of rhesus alloimmunization is difficult to determine. The last year that the U.S. National Center for Health Statistics reported data on the demographic variable rhesus sensitization, which was recorded on birth certificates, was 2003. In that report, 6.8 cases of rhesus alloimmunization were reported per 1,000 live births.1

From the Division of MaternalFetal Medicine, Department of Obstetrics and Gynecology, Baylor College of Medicine; and the Texas Childrens Fetal Center, Texas Childrens Hospital, Houston, Texas. Corresponding author: Kenneth J. Moise Jr, MD, Department of Obstetrics and Gynecology, Division of MaternalFetal Medicine, Baylor College of Medicine, 6620 Main Street, Suite 1100, Houston, TX 77030; e-mail: kjmoise@bcm.edu. Financial Disclosure Dr. Moise is a consultant and received research funding from Sequenom, Inc. (San Diego, CA). 2008 by The American College of Obstetricians and Gynecologists. Published by Lippincott Williams & Wilkins. ISSN: 0029-7844/08

Pathophysiology
Several investigators have reported that spontaneous fetomaternal hemorrhages occur with increasing fre-

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quency and volume with advancing gestational age. Using a sensitive Kleihauer assay, Bowman et al2 noted 0.01 mL or more of fetal cells in 3%, 12%, and 46% of women in each of the three successive trimesters, respectively. In the majority of cases, the antigenic load of rhesus D (RhD) antigen on the fetal erythrocytes and erythrocytic precursors is insufficient to stimulate the maternal immune system. However, in the case of a large antenatal fetomaternal hemorrhage or a fetomaternal hemorrhage at delivery, maternal B lymphocyte clones that recognize the RhD antigen are established. The initial maternal immunoglobulin M anti-D production is short-lived, with a rapid change to an immunoglobulin G response. Memory B lymphocytes then await a new antigenic exposure in the subsequent pregnancy. If stimulated by the RhD antigen on fetal erythrocytes, these plasma cells can then rapidly proliferate and produce immunoglobulin G antibodies and an increase in the maternal titer. Maternal immunoglobulin G crosses the placenta and destroys any RhDpositive erythrocytes, resulting in fetal anemia.

Prevention
Four different rhesus immune globulin products are now available in the United States to prevent rhesus alloimmunization. RhoGAM (Ortho-Clinical Diagnostics, Inc., Rochester, NY) was the first product approved for clinical use; this product has significant name recognition for generic rhesus immune globulin despite the availability of three additional products on the U.S. market. RhoGAM and HyperRHO S/D (Talecris Biotherapeutics-Research Triangle Park, NC) are processed by Cohn cold ethanol fractionation, which results in some contamination with immunoglobulin A antibodies and other plasma proteins. These products must therefore be given by intramuscular injection. The remaining two available rhesus immune globulin preparations (Rhophlac, ABO Pharmaceuticals, Mission Viejo, CA, and WinRho-SDF, Cangene Corporation, Winnipeg, Manitoba, Canada) are prepared by sepharose column and ion-exchange chromatography, respectively. The resulting products contain only immunoglobulin G and therefore can be administered by either the intravenous or intramuscular route. All available products undergo solvent detergent treatment to inactivate enveloped viruses; many manufacturers have added micropore filtration as an additional step to further reduce the chance for viral transmission. Thimerosal, a mercury-based preservative once found in all rhesus immune globulins, is no longer included in the manufacturing process.

Although once produced from the plasma of sensitized women, the decreasing prevalence of rhesus disease has necessitated that the plasma source for rhesus immune globulin be derived from RhD-negative male donors who undergo repeated injections of RhD-positive red cells. Several monoclonal anti-D antibodies are now in clinical trials and may soon replace the current plasma-derived polyclonal products.3 Because rhesus immune globulin is a blood derivative, all patients should be informed of its source and should give informed consent. Although the majority of rhesus immune globulin is issued from hospital blood banks, various manufacturers products can be purchased by private physicians for use in their offices. A recall by one manufacturer in the late 1990s due to inadequate doses in the prefilled syringes warrants that careful records of lot numbers be documented in the patients medical chart and a general clinic logbook. All pregnant patients should undergo an antibody screen at the first prenatal visit. Patients who are determined to be weak rhesus positive (previously Du-positive) are not at risk for rhesus alloimmunization and therefore do not require rhesus immune globulin. If there is no evidence of anti-D alloimmunization in the RhD-negative woman, 300 micrograms of rhesus immune globulin should be administered intramuscularly at 28 weeks of gestation. This practice has been reported to reduce the incidence of antenatal alloimmunization from 2% to 0.1%.4 The American Association of Blood Banks recommends that a repeat antibody screen be obtained before antenatal rhesus immune globulin, although the incidence of alloimmunization before 28 weeks is very low. The cost-effectiveness of this practice has been questioned by the American College of Obstetricians and Gynecologists.5 Maternal sensitization occasionally does occur by 28 weeks, and by not performing the antibody screen the clinician loses the opportunity to detect the hemolytic disease. It would therefore appear prudent to repeat the antibody screen. The maternal blood sample can be drawn at the same office visit as the rhesus immune globulin injection. Although the administration of the exogenous anti-D eventually will result in a weakly positive titer, this will not occur in the short interval of several hours because of the slow absorption from the intramuscular site. Although there are no data to provide guidance, some experts recommend that a second dose of rhesus immune globulin be given if the patient has not delivered by 40 weeks of gestation. Although not well studied, additional indications for the antepartum administration of rhesus immune

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globulin include spontaneous abortion, elective abortion, ectopic pregnancy, genetic amniocentesis, chorion villus sampling, and fetal blood sampling. A dose of 50 micrograms of rhesus immune globulin is effective until 12 weeks of gestation because of the small volume of red cells in the fetoplacental circulation. From a practical standpoint, most hospitals and offices do not stock this dose of rhesus immune globulin; therefore, a standard dose of 300 micrograms is often given. Evidence for the use of rhesus immune globulin in other scenarios that breach the fetoplacental barrier is lacking. Rhesus immune globulin also should be administered for such events as hydatidiform mole, threatened abortion, fetal death in the second or third trimester, blunt trauma to the abdomen, late amniocentesis for assessment of infection or fetal lung maturity, and external cephalic version. In ongoing pregnancies where rhesus immune globulin is administered in the first or second trimester for one of the above indications, a repeat dose still should be given at 28 weeks of gestation. Alternatively, if the antenatal dose was given in the late second trimester (example: 22 weeks for suspected placental abruption), the dose should be repeated 12 weeks later (at 34 weeks of gestation in the example scenario). Because the half-life of rhesus immune globulin is approximately 24 days, 1520% of patients receiving it at 28 weeks will have a very low anti-D titer (usually 2 or 4) detected at the time of admission for labor at term.5 Three hundred micrograms of rhesus immune globulin should be administered within 72 hours of delivery if umbilical cord blood typing reveals an RhD-positive infant. This is sufficient to protect from sensitization caused by a fetomaternal hemorrhage of 30 mL of fetal whole blood. If rhesus immune globulin is inadvertently omitted after delivery, some protection has been proven with administration within 13 days; recommendations have been made to administer it as late as 28 days after delivery.6 If delivery occurs less than 3 weeks from the administration of rhesus immune globulin used for antenatal indications such as amniocentesis for fetal lung maturity or external cephalic version, a repeat dose is unnecessary unless a large fetomaternal hemorrhage is detected at the time of delivery. Despite the widespread acceptance of similar guidelines, studies from Scotland have revealed that two thirds of rhesus alloimmunized cases are secondary to antepartum sensitization; an additional 13% are due to failure to administer rhesus immune globulin for the usual obstetric indications.7

Approximately 1 in 1,000 deliveries will be associated with an excessive fetomaternal hemorrhage; risk factors will identify only 50% of these. Routine screening of all women at the time of delivery for excessive fetomaternal hemorrhage should therefore be undertaken. A qualitative yet sensitive test for fetomaternal hemorrhage, the rosette test, can be performed first. Results return as positive or negative. A negative result warrants administration of a standard dose of rhesus immune globulin. If the rosette is positive, a Kleihauer-Betke stain or fetal cell stain using flow cytometry is undertaken. The percentage of fetal blood cells is multiplied by a factor of 50 to estimate the volume of the fetomaternal hemorrhage. Because this calculation includes an inaccurate estimation of the maternal blood volume, the blood bank will typically indicate that additional vials of rhesus immune globulin should be administered over the calculated amount. No more than five units of rhesus immune globulin should be administered by the intramuscular route in one 24-hour period. This recommendation is based on the number and volume of injections that are required to administer this amount of rhesus immune globulin using prefilled syringes. Should a large dose of rhesus immune globulin be necessary, an alternative method would be to give the entire calculated dose intravenously in divided increments (maximum dose for each increment: 3,000 international units or 600 micrograms) every 8 hours. The administration of rhesus immune globulin after a postpartum tubal ligation is controversial. The possibility of a new partner in conjunction with the availability of in vitro fertilization would seem to make the use of rhesus immune globulin in these situations prudent. In addition, RhD sensitization would limit the availability of blood products if the patient later required a transfusion. Rhesus immune globulin is not effective once alloimmunization to the RhD antigen has occurred.

Diagnostic Approach Genetics

In 1946, Fisher and Race proposed the concept of three genes that encode for the three major rhesus antigen groupsD, C/c, and E/e. Some 45 years later, the rhesus locus was localized to the short arm of chromosome one.8 Only two genes were identifiedan RHD gene and an RHCE gene. Production of two distinct proteins from the latter gene probably occurs as a result of alternate messenger ribonucleic acid splicing. A single cytosine to guanine change in

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Table 1. Incidence of Paternal Heterozygosity (%) Based on Serology, Ethnic Background, and Number of Previous Rhesus D-Positive Offspring
Number of RhD Infants
DCce DCe DCEe DcE DCcEe Dce

White 0
90 9 90 13 11 94

Black 4
36 0.6 36 0.9 0.8 50

Hispanic 4
4 1 4 0.1 0.3 7

1
82 5 82 7 6 89

2
69 2 69 4 3 80

3
53 1 53 2 2 66

5
22 0.3 22 0.5 0.4 33

0
41 19 37 1 10 54

1
26 11 23 0.5 5 37

2
15 6 13 0.3 1 23

3
8 3 7 0.1 0.7 13

5
2 0.7 2 0 12 4

0
85 5 85 2 6 92

1
74 2 74 0.9 3 85

2
59 1 59 0.5 2 74

3
42 0.6 42 0.2 0.8 59

4
26 0.3 26 0.1 0.4 42

5
15 0.1 15 0.1 26

RhD, rhesus D positive. Moise KJ, Jr. Management of rhesus Alloimmunization in Pregnancy. Obstet Gynecol 2002;100:60011.

exon 5 of the RHCE gene results in formation of the e antigen instead of the E antigen.9 One nucleotide difference (cytosine to thymine) in exon 2 of the RHCE gene results in a single amino acid change of a serine to proline. This causes the expression of the C antigen as opposed to the c antigen.10 These discoveries have resulted in major changes in the management of the RhD-sensitized pregnancy. Approximately 55% of individuals are heterozygous at the RHD locus. In these cases, only 50% of their offspring will be RhD positive and have the potential to be affected by the maternal antibody. Alternatively, if the fetus is determined to be RhD negative, no further testing is warranted. Because the RHC/c and E/e genes are inherited in a closely linked fashion to the RHD gene, blood banks can employ antisera to the corresponding antigens along with gene frequency tables based on ethnicity to estimate the chance of a paternal heterozygous state at the RHD locus (see Table 1). As an example, a Caucasian partner who undergoes serologic testing with the following resultsanti-D: positive, anti-C: negative, anti-c: positive, anti-E: negative, and anti-e: positivewould be considered to exhibit the Dce phenotype. If he has never fathered an RhD-positive child in the past, his chance of being heterozygous for RHD would be 94%. A history of previous RhD-positive children fathered by this individual results in a statistical decrease in the chance that he is heterozygous. Finally, a history of an RhD-negative offspring confirms a paternal heterozygous state. More recently, DNA techniques can be employed to accurately determine whether there is one or two paternal RHD genes present. Polymerase chain reaction (PCR) signal intensities of exons 5 and 7 of the RHD gene are compared with an internal control gene, exon 7 of the RHCE gene, to determine the heterozygous or homozygous paternal state.11

Once a heterozygous paternal genotype is confirmed by history (previous history of an RhD-negative offspring) or direct paternal testing through serology or DNA testing, the next step in the evaluation of the sensitized patient should be to determine the RhD type of the fetus (Fig. 1). Chorion villus biopsy has been suggested as another method to obtain fetal DNA for rhesus testing. However, the use of chorion villus biopsy for this purpose should be discouraged because disruption of the chorionic villi during the procedure can result in fetomaternal hemorrhage and an anamnestic response in maternal titer, thereby worsening the fetal disease.12 Fetal DNA can be readily obtained through amniocentesis, however every attempt should be made to avoid transplacental passage of the needle so as to prevent enhanced maternal sensitization. The overall sensitivity and specificity of PCR typing on amniotic fluid has been reported to be 98.7% and 100%, respectively, and the positive and negative predictive values were 100% and 96.9%.13 Inconsistencies can occur due to erroneous paternity or a rearrangement of the paternal RHD gene locus; the latter occurs in up to 2% of individuals. Checking paternal blood (the source of the fetal RHD gene) with the same primers used on the amniotic fluid verifies that a gene rearrangement is not a potential source of error. For this reason, most laboratories offering fetal red cell antigen typing on amniotic fluid require an accompanying paternal blood sample. A situation where paternity is unknown or the patients partner is unavailable does not allow for such confirmation. A repeat maternal anti-D titer obtained after 4 to 6 weeks can serve as a confirmatory strategy. A fourfold rise in titer (ie, 16 to 64) should make the clinician suspect that the RhDnegative fetal result may be erroneous. An RHD pseudogene has been described in 69% of South African blacks and 21% of African Ameri-

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Fig. 1. Algorithm for determining the fetal RhD status in the case of a heterozygous paternal genotype. RHD, rhesus D; PCR, polymerase chain reaction. Modified from Obstet Gynecol 2002;100:600 11, and from Queenan JT, editor. High-risk pregnancy. Washington, DC: ACOG; 2007.
Moise. Rhesus Alloimmunization in Pregnancy. Obstet Gynecol 2008.

cans.14 In this situation, the pregnant patient is RhD negative on serologic testing, but the entire RHD gene is present on her chromosomes. Because the fetus inherits one of its RHD genes from its mother, amniotic PCR testing would, therefore, yield a falsepositive resultthe fetus is RhD negative by serology but RHD positive by genotype. This could lead to unnecessary intervention with its inherent risks. For this reason, a maternal blood sample should also accompany the amniotic fluid sent for fetal RHD testing in an effort to rule out the presence of a maternal RHD pseudogene. If the maternal sample is

positive for this variant, fetal testing for the gene also should be undertaken (Fig. 1). The newest technology to assist in the determination of the fetal RhD type in the case of heterozygous paternity uses free fetal DNA in the maternal circulation. Based on a concept that cell-free tumor DNA could be found in the peripheral circulation of patients with cancer, Lo et al15 were the first to report the presence of the RHD gene in the plasma of women pregnant with an RhD-positive fetus. Further studies have indicated that free fetal DNA can be found in the maternal circulation as early as 38 days of gestation;

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by term it comprises 6% of the total circulating DNA pool. Because apoptosis of placental trophoblasts is the source of this DNA, a relatively short half-life (measured in minutes) has been noted. This makes it an attractive source for prenatal diagnosis because there should not be any residual fetal DNA from previous gestations. In a recent meta-analysis, Geifman-Holtzman et al16 compiled reports of more than 2,000 patients where this technique had been used to determine the fetal RHD status in maternal plasma; an accuracy of 97% was found. Until recently, fetal RHD typing was available only in the United Kingdom and Europe. A reverse transcriptase PCR test is now available in the United States for clinical use.17 Unfortunately, the current assay is inconclusive in approximately 14% of casesthose where there is an RHD-negative female fetus detected. In these cases, the absence of the RHD gene in the fetus cannot be verified because there is no internal control to confirm the presence of free fetal DNA in the specimen. As this methodology improves in the next few years, free fetal DNA typing of the fetus will clearly replace amniocentesis.

mL. A threshold value of 15 international units/mL has been recommended for invasive testing because only mild hemolytic disease of the fetus/newborn is usually noted with anti-D levels below this level.18

Middle Cerebral Artery Doppler

Many investigators have sought to use various ultrasound parameters to allow the noninvasive detection of fetal anemia in cases of rhesus alloimmunization. Unfortunately, parameters such as hydramnios, pericardial effusion, diameter of the umbilical vein, and placental thickness failed to predict the anemic fetus. Hydrops fetalis can be detected easily by ultrasonography, but unfortunately this is a late sign of fetal anemia when hemoglobin values have declined to more than 7 g/dL below the norm for gestational age.19 Previous animal studies have indicated that blood velocities in the anemic fetus are elevated because of low viscosity and increased cardiac output. Mari et al20 were the first to propose that Doppler assessment of the peak velocity in the fetal middle cerebral artery (MCA) could determine accurately whether the human fetus was anemic. In this study, a value more than 1.5 multiples of the median (MoM) for gestational age detected all cases of moderate to severe anemia with a false-positive rate of only 12%. Several small observational studies followed. Divakaren et al21 undertook a meta-analysis of published studies using ultrasonography to detect fetal anemia in alloimmunized pregnancies. The authors found that the diagnostic test with the highest methodological quality was the peak MCA velocity (positive likelihood ratio 8.45, 95% confidence interval 4.7 15.6, negative likelihood ratio 0.02, 95% confidence interval 0.001 0.25). More recently, investigators in the Diagnostic Amniocentesis or Non-invasive Doppler study prospectively assessed the peak MCA velocity and compared it with the gold standard for the detection of fetal anemiaamniocentesis for OD450 analysis.22 One hundred sixty-five fetuses were studied at 10 referral centers; 45% of this study population was found to have severe fetal anemia at cordocentesis. Middle cerebral artery Doppler was noted to have a sensitivity of 88%, a specificity of 82%, and an accuracy of 85%. Doppler outperformed OD450 analysis using the Liley curve, with a 9% improvement in the accuracy. The authors proposed that the use of Doppler alone would have averted more than 50% of the invasive procedures in their series. Technically, the determination of the peak MCA velocity is relatively straightforward. The anterior wing of the sphenoid bone at the base of the skull is located; color or power Doppler then is used to locate

Maternal Titer
The maternal titer is the first step in the evaluation of the RhD-sensitized patient. Old methodologies using albumin or saline should no longer be employed. The human antiglobulin titer (indirect Coombs) is used to determine the degree of alloimmunization. By convention, titer values are reported as the integer of the greatest tube dilution with a positive agglutination reaction (ie, a titer of 16 is equivalent to a dilution of 1:16). Variation in results between laboratories is not uncommon because many commercial laboratories use enhancing media so as to not miss low titer samples. However, in the same laboratory, the titer should not vary by more than one dilution. Thus, an initial titer of 8 that returns 16 may not represent a true increase in the amount of antibody in the maternal circulation. A critical titer is defined as the titer associated with a significant risk for fetal hydrops. When this is present, further testing with more invasive techniques is required. This titer will vary between institutions based on the correlation with clinical outcome of hemolytic disease of the fetus/ newborn. Most centers, however, will not have an adequate number of pregnancies complicated by hydrops fetalis to determine a critical titer; a critical value for anti-D between 8 and 32 is usually used. In Europe and the United Kingdom, the amount of circulating anti-D is compared with an international standard and reported in international units/

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80 70 60 50 40 30 20 20 25 30 Gestational age (weeks) 35 A zone moderate to severe anemia B zone mild anemia C zone normal hematocrit A B C Median 1.5 MoMs 1.29 MoMs

Fig. 4. Middle cerebral artery (MCA)Doppler graph based on gestational age. Modified from Obstet Gynecol 2002; 100:600 11, and from Queenan J, editor. High-risk pregnancy. Washington (DC): American College of Obstetricians and Gynecologists; 2007.
Moise. Rhesus Alloimmunization in Pregnancy. Obstet Gynecol 2008.

Fig. 2. Obtaining a middle cerebral artery Doppler peak systolic velocity. Illustration: John Yanson.
Moise. Rhesus Alloimmunization in Pregnancy. Obstet Gynecol 2008.

the MCA vessel (Figs. 2 and 3). The angle of insonation is maintained on the maternal abdomen to approximate the angle of insonation as close to zero as possible. The MCA vessel closer to the maternal abdominal wall usually is studied, although the posterior vessel will give equivalent results.23 Angle cor-

Fig. 3. Middle cerebral artery (MCA) Doppler using color flow to locate the vessel. Arrow notes the proper location for the pulsed Doppler gate on the MCA vessel closest to the transducer.
Moise. Rhesus Alloimmunization in Pregnancy. Obstet Gynecol 2008.

rection software is usually unnecessary. The Doppler gate then is placed in the proximal MCA as the vessel arises from the carotid siphon because measurements in the more distal aspect of the vessel will be inaccurate due to reduced peak velocities. Finally, the fetus should be in a quiescent state during the Doppler examination because accelerations of the fetal heart rate can result in a false elevation in the peak velocity, especially late in the third trimester.24 Measurements can be initiated as early as 18 weeks of gestation and should be repeated at 1-week to 2-week intervals. Because the normal peak MCA Doppler velocity increases with advancing gestational age, data should be adjusted for the time in pregnancy when the study is undertaken. This can be achieved by plotting the value on a standardized graph (Fig. 4). Alternatively, web-based calculators such as the one found at www. perinatology.com can be used to convert the actual peak MCA velocity to MoMit then can be expressed in a fashion similar to second trimester serum alpha-fetoprotein. After 35 weeks of gestation, there appears to be a higher false-positive rate in the detection of fetal anemia.25 Therefore, late in gestation, a normal MCA velocity can be followed with repeat measurements. If an elevated value is noted, amniocentesis for OD450 and fetal lung maturity testing should be undertaken to determine whether delivery is indicated.

Amniocentesis
For the past 50 years, pregnancies complicated by RhD alloimmunization have been followed using the OD450 to measure the level of bilirubin in the

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Queenan curve necessitates investigation by fetal blood sampling. After 37 weeks of gestation, fetal lung maturity testing can be assessed because induction of labor with mature studies can be considered in lieu of subsequent amniocentesis.

Fetal Blood Sampling

Ultrasound-directed fetal blood sampling (also called percutaneous umbilical blood sampling, cordocentesis, and funipuncture) allows direct access to the fetal circulation to obtain important laboratory values such as hematocrit, direct Coombs, fetal blood type, reticulocyte count, and total bilirubin. Because fetal blood sampling is associated with a 12% rate of fetal death, this procedure is reserved for the detection of fetal anemia once the peak MCA Doppler had exceeded 1.5 MoM. When used in this context, blood should be available for intravascular intrauterine transfusion if fetal anemia is detected (hematocrit less than 30% or less than two standard deviations for gestational age).

Fig. 5. Queenan curve for OD450 values. Rh, rhesus. Published in Queenan JT, Tomai TP, Ural SH, King JC. Deviation in amniotic fluid optical density at a wavelength of 450 nm in Rh-immunized pregnancies from 14 to 40 weeks of gestation: a proposal for clinical management. Am J Obstet Gynecol 1993;168:1370 6.
Moise. Rhesus Alloimmunization in Pregnancy. Obstet Gynecol 2008.

SUMMARY OF CLINICAL MANAGEMENT

amniotic fluid, an indirect indicator of the degree of fetal hemolysis. The original Liley curve was divided into three zones and provided values to be used from 27 weeks of gestation to term. Later, Queenan proposed a modified curve consisting of four zones with data for earlier gestations (Fig. 5). A recent comparative study found the overall sensitivity, specificity, and accuracy of the Queenan curve to be superior to the Liley curve.22 When the two curves were compared for pregnancies at less than 27 weeks of gestation, the sensitivity of the Queenan curve was superior to the Liley curve by 10%; the specificity of both methods was 40%. With the widespread acceptance of serial MCA Dopplers, amniocentesis for OD450 to detect fetal anemia has fallen into disfavor. Because a learning curve is associated with performing MCA Doppler, a center with minimal experience with this technique initially should perform the measurements in conjunction with serial amniocenteses for OD450.26 In addition, serial amniocenteses still may prove useful if a perinatal center skilled in MCA Doppler is not within the patients geographic proximity. If amniocentesis is used to monitor fetal disease, serial procedures are undertaken at 10-day to 2-week intervals and continued until delivery to follow trends in the OD450 values. As mentioned earlier, attempts should be made to avoid transplacental passage of the needle because this can lead to fetomaternal hemorrhage and a rise in maternal antibody titer. A rising or plateauing trend of OD450 values that reaches the upper portion of the Rh-positive affected zone of the The approach using the armamentarium of the various diagnostic tools is based on the maternal history of fetal or neonatal hemolytic disease (Fig. 6). As a general rule, the patients first RhD-sensitized pregnancy involves minimal fetal/neonatal disease; subsequent gestations are associated with a worsening degree of anemia. The rarity of this condition warrants consideration of consultation or referral to a maternalfetal medicine specialist.

First Affected Pregnancy

Once sensitization to the RhD antigen is detected, maternal titers are repeated every month until approximately 24 weeks; titers are repeated every 2 weeks thereafter. Paternal blood is drawn to determine RhD status and zygosity. In cases of a heterozygous paternal phenotype/genotype, once a critical maternal titer of 32 is achieved, an amniotic fluid sample along with maternal and paternal blood samples are sent to a DNA reference laboratory at the time of amniocentesis or free fetal DNA testing to determine the fetal RhD status. In the case of an RhD-negative paternal blood type or fetal RHDnegative genotype, further maternal and fetal monitoring is unwarranted as long as paternity is assured. A homozygous paternal phenotype/genotype indicates an RhD-positive fetus and negates the need for amniocentesis for fetal typing. If there is evidence of an RhD-positive fetus, serial MCA Dopplers are undertaken at 1-week to 2-week intervals starting at around 24 weeks of gesta-

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Fig. 6. Algorithm for the overall management of the pregnant patient with RhD alloimmunization. Rh, rhesus; MCA, middle cerebral artery; MoM, multiple of the median; Hct, hematocrit; EGA, estimated gestational age.
Moise. Rhesus Alloimmunization in Pregnancy. Obstet Gynecol 2008.

tion. An MCA greater than 1.5 MoM at any time between 24 and 35 weeks of gestation is an indication for cordocentesis for fetal hematocrit determination and transfusion as needed. Antenatal testing (nonstress test or biophysical profile) should be initiated after 32 weeks of gestation. If the MCA values remain at 1.5 MoM or less, induction of labor should be considered by 38 weeks of gestation. If a value of more than 1.5 MoM is noted after 35 weeks of gestation, an amniocentesis for OD450 and fetal lung maturity should be performed. If pulmonary maturity is noted and the OD450 is not in the upper Rhpositive affected zone or the intrauterine transfusion

zone of the Queenan curve, induction should be considered at 38 weeks of gestation to allow fetal hepatic maturity to occur. This will decrease the need for prolonged neonatal bililight therapy or the need for exchange transfusion. An amniocentesis result indicating fetal lung maturity and a OD450 in the upper Rh-positive affected zone or the intrauterine transfusion zone indicates the need to proceed with induction of labor. If the amniocentesis indicates fetal pulmonary immaturity in conjunction with a OD450 in the upper Rh-positive affected zone or the intrauterine transfusion zone, one should consider the maternal administration of antenatal steroids. A

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course of maternal oral phenobarbital (30 mg three times daily) also should be considered because this therapy has been shown to reduce the need for neonatal exchange transfusion by up to 75%.27 Induction should occur 1 week later. Finally, if there is fetal lung immaturity and a OD450 value below the upper Rh-positive affected zone, one should consider repeat amniocentesis in 10 days to 2 weeks. Although serial amniocenteses to assess OD450 is less preferred as the primary method of surveillance of the RhD alloimmunized gestation, in those locales in which MCA Doppler testing is not readily available, OD450 values can be assessed in a serial fashion every 10 days to 2 weeks beginning by 24 weeks of gestation. These should be plotted on the Queenan curve, with cordocentesis for possible intrauterine transfusion reserved for those with a OD450 in the upper portion of the rhesus-positive affected zone. At 35 weeks of gestation, fetal lung maturity should be assessed as well and an intervention undertaken according to the above algorithm.

Previously Affected Fetus or Infant

If the patient has a new partner and there is a history of a previous perinatal loss related to hemolytic disease of the fetus/newborn, a previous need for intrauterine transfusion, or a previous need for neonatal exchange transfusion, paternal RhD typing should be undertaken. In the case of an RhD-negative partner and assured paternity, no further testing is necessary. A complicated previous pregnancy with the patients same partner should result in a referral to a perinatal center with experience in the management of the severely alloimmunized pregnancy. In these cases, maternal titers are not predictive of the degree of fetal anemia. In the case of a heterozygous paternal phenotype, amniocentesis or free fetal DNA testing should be performed at 15 weeks of gestation to determine the fetal RhD status. The majority of centers are now using serial MCA Doppler measurements to monitor these pregnancies at risk for fetal hemolytic disease. Testing should begin at 18 weeks of gestation and be repeated every 12 weeks. In the rare case where these pregnancies do not require intrauterine transfusions, the remaining algorithm should be identical to that used in a first affected pregnancy.

THERAPEUTIC APPROACH Intrauterine Transfusion

Historically, the intraperitoneal transfusion remained the mainstay of fetal therapy for almost 20 years after its introduction by Liley in 1963.28 With the advent of

real-time ultrasonography for guidance, direct access to the fetal circulation by puncturing the umbilical cord at its placental insertion became commonplace. As a result, the direct intravascular transfusion has replaced the intraperitoneal transfusion in most centers. Compared with the intraperitoneal transfusion, the intravascular transfusion is clearly advantageous to the hydropic fetus where absorption of cells from the peritoneal cavity is compromised. Some centers continue to incorporate the intraperitoneal transfusion in the form of a combined procedure in conjunction with the intravascular transfusion. This can result in a more stable fetal hematocrit and a more prolonged interval between procedures. Many European centers prefer to use the intrahepatic portion of the umbilical vein as the site of intravascular transfusion. One advantage cited is a lower incidence of fetal bradycardia because an umbilical artery cannot be inadvertently punctured in this location. In addition, extravasated blood can be absorbed from the peritoneal cavity. The red cells for intrauterine transfusion are typically from a blood type O, RhD-negative, cytomegalovirus-negative unit collected in the previous 72 hours. Some centers undertake an extended crossmatch with the maternal blood type to prevent sensitization to new red cell antigens. Cells are packed to a hematocrit of 75 85% to prevent volume overload in the fetus. The unit undergoes 25 Gy of gamma irradiation to prevent fetal graft-versus-host reaction. Additionally, many centers will leukoreduce the unit using millipore filters. This has the theoretical advantage of decreasing cytomegalovirus transmission because this virus usually remains dormant in the polymorphonuclear leukocytes. At the start of the intravascular transfusion procedure, an initial fetal hematocrit is determined by puncture and sampling of the umbilical vein. The optimal site is from the cord near the placental insertion to provide stabilization of the cord. This is not always possible, and sometimes a floating loop of cord must be targeted. A paralyzing agent is usually administered to cause cessation of fetal movement. Our center uses vecuronium at a dose of 0.1 mg/kg of estimated fetal weight. The total amount of red cells to transfuse will depend on the initial fetal hematocrit, fetoplacental blood volume, and hematocrit of the donor unit. If the donor unit has a hematocrit of approximately 75%, the estimated fetal weight in grams using ultrasonography can be multiplied by a factor of 0.02 to determine the volume of red cells to be transfused to achieve a hematocrit increment of 10%.29 A final target hematocrit of 40 50% is used; a decline of approximately 1% per day can be antici-

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pated between transfusions. In the extremely anemic fetus, the initial hematocrit should not be increased by more than fourfold to allow the fetal cardiovascular system to compensate for the acute change in viscosity.30 In this circumstance, a repeat procedure is undertaken 48 hours later to normalize the fetal hematocrit. Hydrops will typically reverse after one or two intravascular transfusions; placentomegaly is the last feature of the hydropic state to reverse. van Kamp et al31 noted that hydrops reversed in 65% of all cases treated with intrauterine transfusions; in cases of severe hydrops, reversal occurred in only 39% of cases. After attainment of a fetal hematocrit of 40 50%, subsequent procedures are scheduled at 14-day intervals until suppression of fetal erythropoiesis is noted on Kleihauer-Betke stains. This usually occurs by the third intrauterine transfusion. Thereafter, the interval for repeat procedures can be determined based on the decline in hematocrit for the individual fetus, usually a 3-week to 4-week interval. An unanticipated rapid decline in fetal hematocrit may reflect bleeding from the umbilical cord puncture site of the previous procedure, transplacental hemorrhage, or maternal sensitization to new red cell antigens to which the fetus is susceptible. Detti et al32 have proposed that the MCA peak systolic velocity may be used to time the second intrauterine transfusion. A modified threshold of 1.32 MoM (instead of 1.5 MoM) should be used to detect moderate-severe anemia. After the second transfusion, there are insufficient data to guide the clinician on the use of MCA Doppler to time subsequent procedures. A calculated decline in hemoglobin of 0.4 g/dL, 0.3 g/dL, and 0.2 g/dL for the first, second, and third transfusion intervals, respectively, can be used to decide when to perform the next intrauterine transfusion.33 During the era of intraperitoneal transfusions, fetuses were routinely delivered at 32 weeks of gestation and often suffered complications of prematurity such as hyaline membrane disease and the need for neonatal exchange transfusions for the treatment of hyperbilirubinemia. As experience with intravascular transfusion became widespread, pregnancies were delivered at later gestational ages. Most authorities now will perform the final intrauterine transfusion at up to 35 weeks of gestation, with delivery anticipated at 3738 weeks.34 Such a practice allows maturation of both the pulmonary and hepatic systems, virtually eliminating the need for neonatal exchange transfusions. After a viable gestational age is attained, performing the transfusion in immediate proximity to the labor and delivery suite appears prudent so that operative delivery can be undertaken if fetal distress

should occur. Finally, the administration of maternal oral phenobarbital may be considered in the 710 days before delivery. This has been proposed to induce hepatic maturity to allow for improved conjugation of bilirubin. One retrospective study has demonstrated a reduction in the need for neonatal exchange transfusions for hyperbilirubinemia.27 The pregnant patient with a history of recurrent early second trimester pregnancy loss due to fetal hemolytic disease is especially challenging. Despite improved ultrasound resolution, targeting an umbilical vessel at less than 20 weeks of gestation can be technically challenging, often leading to iatrogenic fetal death. One approach is to use intraperitoneal intrauterine transfusions as early as 15 to 16 weeks of gestation as a bridging therapy until intravascular transfusions can be undertaken.35 Unfortunately, guidance on the amount of blood to transfuse is lacking. Alternatively, manipulation of the maternal immune system through a combination of serial plasmaphereses followed by weekly intravenous immune globulin also has been reported to delay the need for intrauterine transfusions until later in gestation.36 The authors protocol consisted of a single volume plasmapheresis every other day for three procedures in the 12th week of gestation, with 5% albumin used for volume replacement. The patients immunoglobulin G pool was replaced after the third procedure by administering a 1 g/kg loading dose of intravenous immune globulin (IVIG) diluted in normal saline. The 10% IVIG infusion was started at a rate of 60 mL/h and increased by 30 mL/h every 30 minutes to a maximum rate of 240 mL/h. A second dose of 1 g/kg IVIG was given the following day. The patients then were treated with a weekly dose of 1 g/kg IVIG until 20 weeks of gestation.

Short-Term Outcome
Perinatal survival after intrauterine transfusion varies by center and the experience of the operator. Clearly, intervention before the appearance of hydrops fetalis is preferable. The largest experience at one center is reported from Leiden University in the Netherlands, a national referral center for the entire country. Overall survival in their series of 740 intrauterine transfusions in 254 fetuses was 89%.37 Hydrops fetalis was associated with a decreased rate of survival to 78%.31 In cases of mild hydrops, 98% of fetuses survived; in cases of severe hydrops, only 55% survived. Suppression of erythropoiesis is not uncommon after several intravascular transfusions. These infants are born with a virtual absence of reticulocytes, with their red cell mass almost entirely comprised of donor red cells. Because exchange transfu-

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sion is rarely required, passively acquired maternal antibodies remain in the neonatal circulation for weeks. This results in a 1-month to 3-month period in which the infant may need several top-up red cell transfusions.38 In one series, more than three fourths of transfused fetuses subsequently needed neonatal top-up transfusions.39 Weekly neonatal hematocrit and reticulocyte counts should be assessed. Threshold hematocrit values of less than 30% in the symptomatic infant or less than 20% in the asymptomatic infant have been suggested for transfusion. A neonatal trial with subcutaneous erythropoietin administered three times a week revealed a decreased need for top-up transfusions.40 Iron therapy in these neonates is unnecessary because of elevated stores as a consequence of their in utero hemolytic disease and intrauterine transfusion therapy.

CONCLUSION
Great advances in the treatment of red cell sensitization have markedly diminished perinatal loss. Despite this, an estimated 200 fetuses die each year in the United States secondary to hemolytic disease of the fetus/ newborn or the complications of treatment.20,46 Free fetal DNA testing and serial MCA Doppler now have replaced amniocentesis to identify the at-risk fetus. In the future, maternal immunotherapy may supplant intrauterine transfusion as the standard treatment. REFERENCES
1. Martin JA, Hamilton BE, Sutton PD, Ventura SJ, Menacker F, Munson ML. Births: Final data for 2003. Natl Vital Stat Rep 2005;54:1116. 2. Bowman JM, Pollock JM, Penston LE. Fetomaternal transplacental hemorrhage during pregnancy and after delivery. Vox Sang 1986;51:11721. 3. Sibe ril S, de Romeuf C, Bihoreau N, Fernandez N, Meterreau JL, Regenman A, et al. Selection of a human anti-RhD monoclonal antibody for therapeutic use: impact of IgG glycosylation on activating and inhibitory Fc gamma R functions. Clin Immunol 2006;118:1709. 4. Bowman JM. The prevention of Rh immunization. Transfus Med Rev 1988;2:12950. 5. Prevention of RhD alloimmunization. ACOG Practice Bulletin No. 4. American College of Obstetricians and Gynecologists. Obstet Gynecol 1999. 6. Bowman JM. Controversies in Rh prophylaxis. Who needs Rh immune globulin and when should it be given? Am J Obstet Gynecol 1985;151:28994. 7. Hughes RG, Craig JI, Murphy WG, Greer IA. Causes and clinical consequences of Rhesus (D) haemolytic disease of the newborn: a study of a Scottish population, 19851990. Br J Obstet Gynaecol 1994;101:297300. 8. Che rif-Zahar B, Matte i MG, Le Van Kim C, Bailly P, Cartron JP, Colin Y. Localization of the human Rh blood group gene structure to chromosome region 1p34.3-1p36.1 by in situ hybridization. Hum Genet 1991;86:398400. 9. Carritt B, Kemp TJ, Poulter M. Evolution of the human RH (rhesus) blood group genes: a 50 year old prediction (partially) fulfilled [published erratum appears in Hum Mol Genet 1997; 6:1390]. Hum Mol Genet 1997;6:84350. 10. Avent ND. Fetal genotyping. In: Hadley A, Soothill P, editors. Alloimmune disorders in pregnancy anaemia, thrombocytopenia, and neutropenia in the fetus and newborn. Cambridge (UK): Cambridge University Press; 2002. 11. Pirelli K, Pietz B, Johnson S, Pinder H, Bellissimo D. Molecular determination of RhD zygosity. Am J Obstet Gynecol 2006;195:S172. 12. Moise KJ, Carpenter RJ. Increased severity of fetal hemolytic disease with known rhesus alloimmunization after first-trimester transcervical chorionic villus biopsy. Fetal Diagn Ther 1990;5:768. 13. Van den Veyver IB, Moise KJ. Fetal RhD typing by polymerase chain reaction in pregnancies complicated by rhesus alloimmunization. Obstet Gynecol 1996;88:10617. 14. Singleton BK, Green CA, Avent ND, Martin PG, Smart E, Daka A, et al. The presence of an RHD pseudogene containing a 37 base pair duplication and a nonsense mutation in Africans

Long-Term Outcome
Advances in treatment techniques for the fetus with severe hemolytic disease now allow the more moribund and severely anemic fetus to survive. Standardized developmental assessments at 2 to 3 years of age in children born after successful intrauterine transfusions have failed to detect an association between poor scores and the degree of fetal anemia or the presence of hydrops.41 In one recent series of 16 hydropic patients who survived to 10 years of age, two of the children were found to exhibit severe neurologic morbidity (12.5%).42 Cerebral palsy and developmental delay seem therefore to be more common in fetuses with hemolytic disease of the fetus/newborn when compared with unaffected infants, although a normal outcome can be expected in more than 90% of cases.43 Sensorineural hearing loss is more frequent in these infants, probably because of their prolonged exposure to elevated levels of bilirubin and its toxic effect on the developing eighth cranial nerve.41 Hearing tests should be conducted at birth and yearly for the first few years of life.

Future Treatment
Clearly, selective maternal immunomodulation will be the most promising next breakthrough for the treatment of severe hemolytic disease of the fetus/ newborn. Purposeful immunization to paternal leukocytes has been demonstrated to exhibit a protective effect in the prevention of fetal anemia in a rabbit model.44 Investigation in a transgenic mouse model has indicated that the intranasal administration of specific RhD epitope peptides could be used to ameliorate an established anti-D response, thereby preventing severe hemolytic disease of the fetus/ newborn in a subsequent pregnancy.45

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31. van Kamp IL, Klumper FJ, Bakkum RS, Oepkes D, Meerman RH, Scherjon SA, et al. The severity of immune fetal hydrops is predictive of fetal outcome after intrauterine treatment. Am J Obstet Gynecol 2001;185:66873. 32. Detti L, Oz U, Guney I, Ferguson JE, Bahado-Singh RO, Mari G, et al. Doppler ultrasound velocimetry for timing the second intrauterine transfusion in fetuses with anemia from red cell alloimmunization. Am J Obstet Gynecol 2001;185:104851. 33. Scheier M, Hernandez-Andrade E, Carmo A, Dezerega V, Nicolaides KH. Prediction of fetal anemia in rhesus disease by measurement of fetal middle cerebral artery peak systolic velocity. Ultrasound Obstet Gynecol 2004;23:4326. 34. Klumper FJ, van Kamp IL, Vandenbussche FP, Meerman RH, Oepkes D, Scherjon SA, et al. Benefits and risks of fetal red-cell transfusion after 32 weeks gestation. Eur J Obstet Gynecol Reprod Biol 2000;92:916. 35. Howe DT, Michailidis GD. Intraperitoneal transfusion in severe, early-onset Rh isoimmunization. Obstet Gynecol 2007;110: 8804. 36. Ruma MS, Moise KJ, Kim E, Murtha AP, Prutsman WJ, Hassan SS, et al. Combined plasmapheresis and intravenous immune globulin for the treatment of severe maternal red cell alloimmunization. Am J Obstet Gynecol 2007;196:138.e16. 37. van Kamp IL, Klumper FJ, Oepkes D, Meerman RH, Scherjon SA, Vandenbussche FP, et al. Complications of intrauterine intravascular transfusion for fetal anemia due to maternal red-cell alloimmunization. Am J Obstet Gynecol 2005;192:1717. 38. Saade GR, Moise KJ, Belfort MA, Hesketh DE, Carpenter RJ. Fetal and neonatal hematologic parameters in red cell alloimmunization: predicting the need for late neonatal transfusions. Fetal Diagn Ther 1993;8:1614. 39. De Boer I P, Zeestraten E C, Lopriore E, van Kamp IL, Kanhai HH, Walther FJ. Pediatric outcome in Rhesus hemolytic disease treated with and without intrauterine transfusion. Am J Obstet Gynecol 2008;198:54 e14. 40. Ovali F, Samanci N, Dag og lu T. Management of late anemia in Rhesus hemolytic disease: use of recombinant human erythropoietin (a pilot study). Pediatr Res 1996;39:8314. 41. Hudon L, Moise KJ Hegemier SE, Hill RM, Moise AA, Smith EO, et al. Long-term neurodevelopmental outcome after intrauterine transfusion for the treatment of fetal hemolytic disease. Am J Obstet Gynecol 1998;179:85863. 42. Harper DC, Swingle HM, Weiner CP, Bonthius DJ, Aylward GP, Widness JA. Long-term neurodevelopmental outcome and brain volume after treatment for hydrops fetalis by in utero intravascular transfusion. Am J Obstet Gynecol 2006;195:192200. 43. Moise KJ, Whitecar PW. Antenatal therapy for haemolytic disease of the fetus and newborn. In: Hadley A, Soothill P, editors. Alloimmune disorders in pregnancy anaemia, thrombocytopenia and neutropenia in the fetus and newborn. Cambridge (UK): Cambridge University Press; 2002. 44. Whitecar PW, Farb R, Subramanyam L, Dorman K, Balu RB, Moise KJ. Paternal leukocyte alloimmunization as a treatment for hemolytic disease of the newborn in a rabbit model. Am J Obstet Gynecol 2002;187:97780. 45. Hall AM, Cairns LS, Altmann DM, Barker RN, Urbaniak SJ. Immune responses and tolerance to the RhD blood group protein in HLA-transgenic mice. Blood 2005;105:21759. 46. Schumacher B, Moise KJ. Fetal transfusion for red blood cell alloimmunization in pregnancy. Obstet Gynecol 1996;88: 13750.

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