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Red Blood Cell Alloimmunization in Pregnancy
Red Blood Cell Alloimmunization in Pregnancy Kenneth J. Moise Jr Red blood cell (RBC) alloimmunization in pregnancy continues to occur despite the widespread use of both antenatal and postpartum Rhesus immune globulin (RhIG), due mainly to inadvertent omissions in administration as well as antenatal sensitization prior to RhIG given at 28 weeks’ gestation. Additional instances are attributable to the lack of immune globulins to other RBC antigens. Evaluation of the alloimmunized pregnancy begins with the maternal titer. Once a critical value [32 for anti-Rh(D) and other irregular antibodies; 8 for anti-K and – k] is reached, fetal surveillance using serial Doppler ultrasound measurements of the peak velocity in the fetal middle cerebral artery (MCA) is standard. In the case of a heterozygous paternal phenotype, amniocentesis can be performed to detect the antigen-negative fetus that requires no further evaluation. MCA velocities greater than 1.5 multiples of the median necessitate cordocentesis, and if fetal anemia is detected, intrauterine transfusion therapy is initiated. A perinatal survival of greater than 85% with normal neurologic outcome is now expected. Future therapies will target specific immune manipulations in the pregnant patient. Semin Hematol 42:169-178 © 2005 Elsevier Inc. All rights reserved.
H
istorically, the fetal effects of maternal red blood cell (RBC) alloimmunization were undetectable until after the birth of an affected infant. These neonatal sequelae of maternal RBC alloimmunization became known as hemolytic disease of the newborn (HDN) or erythroblastosis fetalis. As a consequence of the declining rate of RhD alloimmunization secondary to the success of Rhesus immune globulin (RhIG), patients with more severe disease should be referred to a maternal-fetal medicine physician (formerly known as a perinatologist). These subspecialty-boarded individuals have completed a 2- or 3-year fellowship training program in highrisk obstetrics after a standard 4-year residency training period in obstetrics and gynecology. In the skilled hands of the maternal-fetal medicine physician, obstetrical ultrasound and direct access to the fetal circulation by cordocentesis has made the detection of fetal anemia a reality. Therefore it is now appropriate to refer to the perinatal effects of maternal RBC alloimmunization as hemolytic disease of the fetus and newborn (HDFN).
Significance and Incidence of RBC Antibodies More than 50 different RBC antigens can cause HDFN (Table 1). However, only anti-Rh(D), -Rh(c), and -Kell (K1) commonly appear in large case series of severe HDFN treated with intrauterine transfusion (IUT). In a recently published report of 254 fetuses referred to a tertiary care center for IUT in the Netherlands, 85% involved anti-D, 10% anti-K1, and 3.5% anti-c; there was one case each of anti-E, anti-e, and anti-Fya.1 A recent series of 1,133 Dutch women screened positive for antibodies associated with HDFN at 12 weeks of gestation2: anti-E was the most common antibody detected (23%) followed by anti-K (18.8%); anti-D was the third most common antibody detected (18.7%) followed by anti-c (10.4%), anti-C (7.5%), anti-Fya (5.8%), anti-Cw (5.7%), anti-S (3.5%), anti-Jka (2.5%), and miscellaneous other antibodies (3.7%). Current complete birth certificate data analyzed at the Centers for Disease Control found that in the year 2002, approximately 6.7 cases of Rhesus alloimmunization occurred per 1,000 live births in the United States.3
Genetics of the Rhesus Locus Division of Maternal-Fetal Medicine, University of North Carolina School of Medicine, Chapel Hill, NC. Address correspondence to Kenneth J. Moise Jr, MD, Director, Division of Maternal-Fetal Medicine, University of North Carolina School of Medicine, 214 MacNider Bldg CB#7516, Chapel Hill, NC 27599-7516. E-mail: [email protected]
0037-1963/05/$-see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1053/j.seminhematol.2005.04.007
Although three genes originally were proposed to encode for the Rh blood groups, the Rh locus is now known to consist of two genes located on the short arm of chromosome 1.4 Each is 10 exons in length and there is 96% homology between the genes. Production of two distinct proteins from the RhCE 169
K.J. Moise Jr
170 Table 1 Non-Rh(D) Antibodies and Associated HDFN Antigen System
Specific Antigen
Antigen System
Specific Antigen
Antigen System
Specific Antigen
Frequently associated with severe disease Kell Rh
-K (K1) -c
Colton
-Coa -Co3 -ELO -Dia -Dib -Wra -Wrb -Fya -Jsb -k (K2) -Kpa -Kpb -K11 -K22 -Ku -U1a -Jka -Ena -Far -Hil -Hut -M -Mia -Mta -MUT
Diego
Duffy Kell
Kidd MNS
Duffy Gerbich
Kell
-Fyb -Fy3 -Ge2 -Ge3 -Ge4 -Lsa -Jsa
Infrequently associated with severe disease MNS -Mur -MV -s -sD -S -U -Vw Rh -Bea -C -Ce -CW -ce -E -EW -Evans -G -Goa -Hr -Hro -JAL -Rh32 -Rh42 -Rh46 -STEM -Tar Associated with mild disease Kidd -Jkb -Jk3 MNS -Mit Rh -CX -DW -e -HOFM -LOCR
Scianna Other Ag’s
Rh Other
-Sc2 -Rd -Bi -Good -Heibel -HJK -Hta -Jones -Joslin -Kg -Kuhn -Lia -MAM -Niemetz -REIT -Reiter -Rd -Sharp -Vel -Zd
-Riv -RH29 -Ata -JFV -Jra -Lan
Reproduced with permission from Moise KJ: Hemolytic disease of the fetus and newborn, in Creasy RK, Resnik R, Iams J (eds): Maternal-Fetal Medicine, Principles and Practice (ed 5). Philadelphia, PA, Elsevier, Copyright © 2004.
gene probably occurs as a result of alternative splicing of messenger RNA.5 Further study of the RHD gene has revealed significant heterogeneity. Several of these genetic modifications result in a lack of expression of the Rh(D) phenotype. One such example is the RHD pseudogene, which has been found in 69% of South African blacks and 24% of African-Americans.6 All 10 exons of the RHD gene are present, but transcription of the gene into a messenger RNA product does not occur due to the presence of a stop codon in the intron between exons 3 and 4. Similarly, the Ccdes gene has been detected in 22% of African-Americans; it appears to contain exons 1, 2, 9, and 10 as well as a portion of exon 3 of the original RH(D) gene, with other exons being duplicated from the RhCE gene. The presence of these genes in an Rh(D)-negative pregnant patient has
important implications for the prenatal diagnosis of the fetal blood type (see below).
Diagnostic Methods Maternal Antibody Determination Once a maternal antibody screen reveals the presence of anti-D, an antiglobulin titer is the first step in the evaluation of the RhD-sensitized patient during her first affected pregnancy. Sera from each titer should be frozen and used for determining subsequent values, in tandem for purposes of comparison. Many patients and obstetricians will not realize that a one tube change in dilution does not represent a true increase or decline in the level of antibody. Titers are usually
Red blood cell alloimmunization in pregnancy
171
Table 2 Incidence of Paternal Heterozygosity (%) Based on Serology, Ethnic Background, and Number of Previous RhD-Positive Offspring No. of RhDⴙ Infants Caucasian
DCce DCe DcEe DcE DCcEe Dce
Black
Hispanic
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
90 9 90 13 11 94
82 5 82 7 6 89
69 2 69 4 3 80
53 1 53 2 2 66
36 0.6 36 0.9 0.8 50
22 0.3 22 0.5 0.4 33
41 19 37 1 10 54
26 11 23 0.5 5 37
15 6 13 0.3 3 23
8 3 7 0.1 1 13
4 1 4 0.1 0.7 7
2 0.7 2 0 0.3 4
85 5 85 2 12 92
74 2 74 0.9 6 85
59 1 59 0.5 3 74
42 0.6 42 0.2 2 59
26 0.3 26 0.1 0.8 42
15 0.1 15 0.1 0.4 26
Reproduced with permission from Moise KJ: Modern management of Rhesus alloimmunzation in pregnancy. Obstet Gynecol 100:600 – 611, 2002. Copyright 2002, Lippincott Williams & Wilkins.
repeated at 4-week intervals until the middle of the second trimester (24 weeks’ gestation); thereafter, they are repeated every 2 weeks. A critical titer is defined as the antibody titer associated with a significant risk for hydrops fetalis; when present, further fetal surveillance is warranted. This value will vary with institution and methodologies, but in most centers a critical titer for anti-D of 32 is usually used in the first affected pregnancy. Standard tube methodology should be utilized in determining the critical titer, as newer gel microcolumn assays have been reported to result in titers that are 3.4-fold higher.7 Maternal titers are not useful in clinical management after the first affected pregnancy. The generally recommended critical titer for other irregular RBC antibodies is 32. The notable exceptions are the two antibodies to antigens in the Kell system, anti-K and anti-k, for which a lower critical value of 8 has been suggested for maternal antibody.8
Fetal Blood Typing The initial step in determining the fetal blood type is to establish paternal zygosity. Since the RHC/c and E/e genes are inherited in a closely linked fashion to RHD, antisera for these antigens can be used with gene frequency tables. Ethnicity must be incorporated in to the calculation of the likelihood of heterozygosity, as very disparate results can be obtained with similar serologic findings. In addition, Bayesian analysis can be employed to modify the incidence of heterozyosity based on the paternal history of previous Rh(D)-positive offspring (Table 2).9 When the lines of ethnicity are murky, the use of serology and historic population studies become less reliable means of determining paternal zygosity. Quantitative polymerase chain reaction (PCR) has been used with some accuracy to compare the number of copies of the Rh(D) gene in a particular individual to a baseline control gene such as human albumin.10 In addition, it is now recognized that, in the majority of individuals with a gene deletion as the etiology of their Rh(D)-negative status, there are two silent gene segments flanking the main RHD gene. The loss of the RHD gene results in a combined hybrid of these two Rhesus boxes, which can be detected through restriction fragment length polymorphism analysis.2 Although not currently commer-
cially available, this assay will probably be used in the near future to determine paternal zygosity for RHD. After paternal testing has revealed the possibility of a heterozygous state for RHD, fetal testing is indicated. Chorion villus biopsy (placental aspiration under ultrasound guidance) can be undertaken at 10 to 14 weeks’ gestation to obtain genetic material for detection of the RHD gene. The major disadvantage of this method is that disruption of the chorion villi during the procedure can result in fetomaternal hemorrhage and a subsequent rise in maternal titer thereby worsening the fetal disease.11 This procedure should be discouraged unless the patient plans to electively abort an antigen-positive fetus that is detected. In 1990, amniocentesis was described as a reliable method for assessing the fetal blood type through DNA testing.12 Extensive experience using amniocentesis to determine fetal blood type has revealed rare discrepancies in fetal RHD typing. A false negative result occurs when amniotic fluid analysis finds an RHD-negative fetus that is confirmed to be RHDpositive by serology after birth. In this case, the usual fetal surveillance techniques would not be employed and there would be the potential for perinatal loss. In one series of 500 amniocenteses, false negative results occurred in 1.5% of cases.13 The most likely etiology for discordance is either erroneous paternity or a rearrangement at the paternal RHD gene locus. Such gene rearrangements occur in approximately 2% of individuals14 and thus most laboratories offering fetal RBC typing on amniotic fluid require an accompanying paternal blood sample. Verifying that paternal serology and DNA testing reveal equivalent results confirms that a paternal gene rearrangement is not a potential source for error in fetal testing. Alternatively, the use of multiplex PCR targeting at least two different exons of the RHD gene can decrease the likelihood of nondetection of a gene rearrangement. In situations where paternity is not assured or there is no partner available to contribute a blood sample for confirmation of PCR primers, the result of a RHD-negative fetal blood type from amniotic fluid DNA analysis must be considered suspect. Serial maternal titers in patients with Rh(D) alloimmunization who subsequently delivered Rh(D)-negative off-
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Figure 1 Algorithm for clinical management of a patient with red cell sensitization in the first affected pregnancy. (Modified from Moise, KJ: Rh disease: It’s still a threat. Contemp Obstet Gynecol 49:34-48, 2004.)
spring were noted to rise by fourfold in less than 2% of cases.15 In situations of questionable paternity or lack of availability of a paternal blood sample, a repeat maternal antiglobulin titer obtained at 4 to 6 weeks after the results of the amniocentesis offers confirmation (Fig 1); if a fourfold or greater rise in maternal antibody titer is noted (32 to 128), then an Rh(D)-negative result on amniotic fluid should be questioned. Repeat amniocentesis to evaluate the delta optical density at 450 nm (⌬OD450) or fetal blood sampling to determine the fetal Rh(D) status using serologic techniques may be advisable. If the maternal race is black, then the presence of a maternal pseudogene or Ccdes gene should be considered in the scheme of fetal testing. The presence of one of these genes in the fetus can lead to a false positive diagnosis; the amniotic fluid result would be Rh(D)-positive yet the fetus would be found to be Rh(D)-negative by serology after birth, leading to unnecessary fetal interventions with their inherent risks. For this reason, a maternal blood sample should always accom-
pany the amniotic fluid aliquot sent for fetal RHD testing in an effort to rule out the presence of a maternal RhD pseudogene or Ccdes gene. If the maternal sample is positive for one of these variants, then fetal testing for the gene should also be undertaken. DNA analysis of amniotic fluid for fetal typing has been successfully employed for most major RBC antigens associated with HDFN including Rh(D), Rh(C/c), Rh(E/e), K/k, Jka/Jkb, and Fya/Fyb.12,16-19 Fetal Rh(D) determination through noninvasive testing is now routine in other countries.20 Free fetal DNA is cleared from the maternal plasma within minutes after delivery, thereby eliminating the possibility of contamination from a previous gestation.21 In a series of 893 Rh(D)-negative pregnant patients, 42 samples were noted to exhibit a nonfunctional or rearranged RhD gene. After these were excluded, free DNA was accurate in 99.5% of cases in determining the fetal RhD type.22 Once the issue of an aberrant RH(D) gene has been addressed, RHD-positive results on free DNA can be
Red blood cell alloimmunization in pregnancy considered reliable as RHD-positive genetic material cannot be from a maternal source. A fetal RHD-negative result is more problematic. If fetal DNA fails to amplify in a background of overwhelming maternal DNA in plasma, an RHDnegative result will be obtained. One internal control is the detection of the SRY gene found in association with male fetuses; the presence of this gene in free DNA indicates that fetal DNA is present and an Rh(D)-negative result is reliable.20 For a female fetus, DNA polymorphisms noted in the maternal white blood cells can be used as an internal control.20 If different polymorphisms than those found in the mother are present in the plasma sample, fetal DNA is present. Again, in this situation the finding of an Rh(D)negative fetus can be considered reliable. In as many as 4% of cases, DNA polymorphisms are not informative so the RhDnegative fetal result remains questionable; a repeat maternal sample could be submitted or amniocentesis could be undertaken to determine the fetal RhD type. Only free fetal DNA testing for RHD typing of the fetus is currently performed, although assays for K, RHE, and RHc will be available in the near future.2
Amniocentesis for ⌬OD450 Since first introduced to clinical practice by Bevis,23 spectral analysis of amniotic fluid at 450 nm (⌬OD450) has been used to measure the level of bilirubin, an indirect indicator of the degree of fetal hemolysis. Liley24 proposed a management scheme involving three zones based on gestational ages between 27 and 42 weeks. Zone 1 indicated mild HDFN while zone 3 was associated with fetal death within 10 days. Extrapolated Liley curves to gestational ages earlier than 27 weeks have proven erroneous in diagnosing fetal anemia.25 A modified ⌬OD450 curve for such situations has been published by Queenan et al26: four zones were proposed, with the upper two zones being termed the Rh-positive, affected zone and the intrauterine death zone. Serial amniocenteses have been successfully employed in the past to follow alloimmunized pregnancies to non-Rh(D) antibodies.27,28 The notable exception has been in patients sensitized to Kell antigens. In vitro and in vivo studies have shown that fetal anemia secondary to anti-K is secondary to two mechanisms: hemolysis and erythropoietic suppression.29,30 Amniotic fluid ⌬OD450 values do not reflect the degree of fetal anemia as accurately as in cases involving anti-RhD. As a result some recommend a lower threshold of the 65 percentile of zone 2 of the Liley curve to proceed to cordocentesis.8 Alternatively, middle cerebral artery (MCA) Doppler measurements are probably more accurate than ⌬OD450 values in these cases (see below). 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. All attempts should be made to avoid transplacental passage of the needle, since this can lead to fetomaternal hemorrhage and a rise in maternal antibody titer. A rising or plateauing ⌬OD450 value that reaches the eightieth percentile of zone 2 of the Liley curve or a value that enters the upper
173 portion of the Rh-positive, affected zone of the Queenan curve requires investigation by fetal blood sampling.
Fetal Blood Sampling Ultrasound-directed fetal blood sampling (FBS; also cordocentesis, percutaneous umbilical blood sampling, and funipuncture) allows direct assess to the fetal circulation to obtain important laboratory values such as fetal blood type, hematocrit, direct Coombs, reticulocyte count, and total bilirubin. In this procedure, a 20- or 22-gauge needle is directed into the amniotic cavity under continuous ultrasound guidance. The umbilical cord is targeted at its insertion into the placenta. In the case of an anterior placenta, the needled is directed through the placental mass into the cord root. Although a free-floating cord can be used, the ease of mobility in the liquid environment makes for a more difficult procedure. Procedure-related complications include anamnestic response in maternal antibody titer, fetal exsanguination from the puncture site, transient fetal bradycardia, cord hematoma, and fetal death.31,32 One large series reported a perinatal loss rate of 1% after cordocentesis for diagnostic purposes.33 Although there are three potential vessels in the umbilical cord for vascular access—two arteries and one vein—the umbilical vein represents a larger target and has been associated with a lower incidence of fetal bradycardia as compared to puncture of an umbilical artery.1 For these reasons, cordocentesis is reserved for patients with elevated ⌬OD450 values or elevated peak MCA Doppler velocities (see below).
Ultrasound Perhaps the greatest advance in the management of the alloimmunized pregnancy has been the use of ultrasound. Gestational age can be accurately established in order to evaluate fetal parameters that vary with fetal age, including hemoglobin, ⌬OD450 levels, and peak MCA Doppler velocities. Hydrops fetalis is defined as the presence of extracellular fluid in at least two fetal compartments. Often ascites is the first sign of impending hydrops, with scalp edema and pleural effusions noted with worsening anemia. When hydrops is present, fetal hemoglobin deficits of 7 to 10 g/dL from the mean hemoglobin value for the corresponding gestational age can be expected34— unfortunately, this represents the end-stage state of fetal anemia. Survival with intrauterine transfusion is markedly reduced in these cases, and therefore many investigators have sought alternative ultrasound parameters that could predict the early onset of anemia.35 Although assessment of fetal hepatosplenomegaly using ultrasound has demonstrated some ability to predict fetal anemia, Doppler ultrasound studies of the peak velocity in the MCA have been most predictive (Fig 2).36,37 The severely anemic fetus exhibits an increased cardiac output in an effort to enhance oxygen delivery to peripheral tissues.38 In addition, fetal anemia is associated with a lower blood viscosity, which produces less shearing forces in blood vessels, resulting in increased blood velocities. In a recent study 111 fetuses at risk for fetal anemia secondary to HDFN were assessed using
K.J. Moise Jr
174
Figure 2 Middle cerebral artery Doppler determination; arrows denote anterior and posterior fetal MCA vessels. Dotted line and box denoted angle of insonation and Doppler gate for measurement of the peak velocity.
receiver operator curve analysis.39 A threshold value of 1.5 multiples of the median (MoMs) for the peak MCA velocity was used to predict moderate to severe anemia (⬍0.65 MoMs for fetal hemoglobin). This degree of anemia was predicted with a sensitivity of 100% and a false positive rate of 10%. A recent prospective, multicenter trial confirmed these initial results with a sensitivity of 88% and a negative predictive rate of 89%.40 Many centers have now replaced serial amniocenteses for ⌬OD450 with serial MCA Doppler. MCA measurements can be obtained reliability as early as 18 weeks’ gestation. Studies are repeated every 1 to 2 weeks depending on the trend (Fig 3); values can be plotted on standard curves or converted to MoMs using internet-based calculators, but values after 35 weeks’ gestation are associated with a higher rate of false positives. If MCA Doppler were routinely employed, more than 70% of invasive testing could be avoided using this modality to monitor alloimmunized pregnancies.
Clinical Management The approach using available diagnostic tools is based on the patient’s past history of fetal or neonatal manifestations. The patient’s first antigen-sensitized pregnancy usually involves
Figure 3 Serial MCA Doppler studies in one patient who required intrauterine transfusion. Vertical axis represents the MCA velocity in cm/s; the horizontal axis represents advancing gestational age in weeks. Zone A denotes mild anemia zone; Zone B denotes moderate to severe anemia zone. Cordo, cordocentesis; MoM, multiples of the median. (Reproduced with permission from Moise KJ: Hemolytic disease of the fetus and newborn, in Creasy RK, Resnik R, Iams J (eds): Maternal-Fetal Medicine, Principles and Practice (ed 5): Philadelphia, PA, Elsevier, Copyright © 2004).
Red blood cell alloimmunization in pregnancy clinically minimal HDFN; subsequent gestations are associated with a worsening degree of anemia.
First Affected Pregnancy Once sensitization to a significant RBC antigen is detected, maternal titers are repeated every month until approximately 24 weeks; titers are repeated every 2 weeks thereafter (Fig 1). Paternal blood is drawn to determine antigen status and zygosity through serologic testing. Once a critical maternal titer is reached (usually 32), serial MCA Doppler examinations are initiated at approximately 24 weeks’ gestation and then repeated every 1 to 2 weeks depending on their trend. In remote geographic areas where access to a referral center for MCA Doppler is impractical, serial amniocenteses for ⌬OD450 starting at 24 weeks and repeated at 10-day to 2-week intervals is acceptable. In cases of a heterozygous paternal phenotype, an amniotic fluid sample obtained at the time of the initial amniocentesis accompanied by a maternal and paternal blood sample should be sent to a DNA reference laboratory to determine the fetal antigen status. In the case of an antigen-negative paternal blood type (with assured paternity) or a fetal antigen-negative genotype on amniotic fluid analysis, no further maternal or fetal monitoring is needed. If there is evidence of an antigen-positive fetus (a homozygous paternal phenotype or antigen-positive fetus by PCR testing on amniotic fluid), serial fetal surveillance is indicated. If an MCA Doppler shows ⱖ1.5 MoMs, cordocentesis should be undertaken at an experienced referral center with blood readied for intrauterine transfusion if the fetal hematocrit is found to be less than 30%. Alternatively, if the ⌬OD450 rises into or plateaus in the upper portion of the Rh-positive, affected zone of the Queenan curve or the eightieth percentile of the Liley curve, cordocentesis should be planned. If the peak MCA value remains at less than 1.5 MoMs, amniocentesis at 35 weeks’ gestation should be considered. When fetal pulmonary maturity is present and the ⌬OD450 is not in the upper portion of the Rh-positive, affected zone of the Queenan curve, delivery should be induced in 2 weeks. This time interval enables fetal hepatic maturity to occur and limits the need for exchange transfusion secondary to neonatal hyperbilirubinemia. If fetal pulmonary immaturity is noted at the 35-week amniocentesis and the ⌬OD450 is in the Rh-positive, affected zone of the Queenan curve, administration of maternal phenobarbital (30 mg orally three times per day) with induction 1 week later decreases the probability of later neonatal exchange transfusion.41 Finally, if results indicate fetal lung immaturity with a ⌬OD450 that is not in the Rh-positive, affected zone of the Queenan curve, a repeat amniocentesis should be undertaken in 2 weeks. Management of a pregnancy with serial amniocenteses for ⌬OD450 would follow this same management plan in the late third trimester.
Previously Affected Fetus or Infant If there is a history of a previous perinatal loss related to HDFN, intrauterine transfusion, or neonatal exchange trans-
175 fusion, the patient should be referred to a tertiary care center experienced in the management of the severely alloimmunized pregnancy (Fig 4). In these cases, maternal titers are not predictive of the degree of fetal anemia. When the paternal phenotype is heterozygous, amniocentesis at 15 weeks’ gestation to determine the fetal antigen status is indicated. Serial MCA Doppler measurements should begin at 18 weeks’ gestation and be repeated every 1 to 2 weeks. Alternatively, serial amniocenteses for measurement of ⌬OD450 can be used with the Queenan curve for reference values beginning at 18 weeks. Subsequent surveillance is similar as for the first affected pregnancy.
Intrauterine Transfusion Since it was first introduced in 1963 by William Liley,42 IUT of RBCs has proven lifesaving for cases of severe HDFN. The original approach was to infuse donor RBCs into the peritoneal cavity of the fetus, where they would be subsequently absorbed through the diaphragmatic lymphatics. Today, IUTs are performed as direct infusions of RBCs into the umbilical cord or into the intrahepatic portion of the umbilical vein of the fetus. Some centers continue to use the intraperitoneal approach as part of a combined technique with intravascular transfusion in an effort to create a reservoir of RBCs between procedures.43 Typically a freshly donated cytomegalovirus-negative unit of type O, Rh(D)-negative RBCs is cross-matched to a maternal sample; the unit is leukoreduced, irradiated with 25 Gy to prevent graft-versus-host reaction, and then packed to a final hematocrit of approximately 75% to 80%. A unit with a hematocrit of less than 75% could cause volume overload in the fetus, while a hematocrit of greater than 85% is too viscous and difficult to transfuse through the long, small-diameter procedure needle. The initial step in the procedure is to introduce the needle under continuous ultrasound guidance into the umbilical vein. A sample of fetal blood is obtained for initial hematocrit. Optimally, the sample is processed as a spun hematocrit or through the use of an automated hemocytometer located in the operating room. A short-term paralytic agent such as vecuronium or atracurium is administered to the fetus to cause cessation of fetal movement. The amount of packed RBCs to be infused is based on the estimated fetal weight as determined by ultrasound and standardized formulas.44 A final fetal hematocrit of approximately 40% is targeted. Severely anemic fetuses in the early second trimester do not tolerate acute correction of their hematocrit to normal values,45 and the initial hematocrit should not be increased by more than four-fold at the time of the first procedure.46 A repeat intravascular transfusion is then performed within 48 hours to correct the fetal hematocrit into the normal range. At the conclusion of the procedure, a small aliquot of blood is obtained to measure the hematocrit as well as the percentage of fetal versus adult hemoglobin-containing RBCs through either a Kleihauer-Betke stain or fetal flow cytometry. After the first IUT, subsequent procedures can be empirically scheduled at 14-day intervals until suppression of
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Figure 4 Algorithm for clinical management of a patient with red cell sensitization and a previously affected fetus or infant. (Modified from Moise KJ: Rh disease: It’s still a threat. Contemp Obstet Gynecol 49:34-48, 2004.)
fetal erythropoiesis is observed, usually by the third IUT. Thereafter, the interval for repeat procedures is based on the decline in hematocrit for the individual fetus, usually every 3 to 4 weeks. A final procedure is usually not performed past 35 weeks’ gestation and the patient is scheduled for delivery approximately 3 weeks later.
Outcome Survival after IUT varies by center, experience, and the presence of hydrops fetalis. Overall survival in one review was 84%; up to one fourth fewer hydropic fetuses survive with IUT (70%) as compared to fetuses who undergo their first IUT when they are not hydropic (92%).47 The experience of a single treatment center with 254 fetuses receiving 740 IUTs indicated an overall survival rate of 89%.1 The practice of prolonging the gestation of the treated fetus with HDFN until near term has resulted in a rare necessity for
a neonatal exchange transfusion. Typically, these infants are born with a virtual absence of reticulocytes and a RBC population consisting mainly of transfused blood. The blood bank may be confused if cord blood at delivery is submitted for neonatal RBC typing—the neonate will be typed as O, Rh(D)-negative reflecting the antigen status of the donor blood used for the IUTs! Elevated levels of circulating maternal antibodies in the neonatal circulation in conjunction with suppression of the fetal bone marrow will result in the need for neonatal RBC top-up transfusions at approximately 1 month of age in 50% of these infants.48 These children should be monitored weekly by hematocrits and reticulocyte counts until there is evidence of recovery of hematopoietic function. Only limited data are available for counseling the patient regarding long-term outcome, since fetuses with severe anemia and hydrops are likely to survive today secondary to the use of intravascualr transfusions. Short-term studies at up to
Red blood cell alloimmunization in pregnancy 2 years of age have revealed that more than 90% of infants are neurologically normal despite the history of hydrops fetalis.49,50
Other Treatment Modalities Prior to the advent of the IUT, plasmapheresis represented one of the few therapeutic modalities for severe HDFN. The literature consists mainly of single patients or relatively small series. Despite these limitations, a review of the published cases reveals a perinatal survival rate of 69%.51 Intravenous immune globulin (IVIG) has been used effectively as the sole antenatal treatment for HDFN. In a case-control study of 69 patients, IVIG given prior to 20 weeks’ gestation followed by IUT was compared to the use of only IUTs after 20 weeks.52 Seventy-three percent of the IVIG group and 56% of the IUT group had experienced previous perinatal losses. Hydrops at the first IUT occurred in one fourth of the IVIG/IUT group as compared to three fourths of the IUT-only group. Importantly, the first IUT was performed a median of 1.5 weeks later in gestation in the patients that had received IVIG. Some experts have proposed a combined approach in patients with a previous perinatal loss in the early second trimester, when technical limitations make the success of IUT unlikely.53 Plasmapheresis is started at 12 weeks’ gestation and repeated three times in that week. The maternal titer should be expected to be reduced by 50%. IVIG is then given to replace the globulin fraction as a 2-g/kg loading dose after the third plasmapheresis; this is followed by 1 g/kg/wk of IVIG until 20 weeks’ gestation.
Future Therapeutic Options Patients with high antibody titers and recurrent perinatal loss in the second trimester have few options other than artificial insemination with RBC antigen-negative donor semen, surrogate pregnancy, or preimplantation diagnosis.54 Future therapy will probably involve selective manipulation of the maternal immune system. In vitro data and clinical case reports suggest that maternal alloantibodies to paternal leukocytes may result in an Fc blockade, thereby protecting the fetal RBCs from hemolysis in cases of Rh(D) alloimmunization. In a rabbit model for HDFN, alloimmunization to paternal leukocytes resulted in fetal hemoglobin levels that approached normal in does that had been previously sensitized to RBCs.55 As an alternative strategy, four peptides have been associated with the proliferation of T-helper cells involved in the development of antibody to the RhD antigen; therapeutic administration of these peptides might ameliorate an established anti-D response, preventing severe HDFN in a subsequent pregnancy.56
Conclusion Major advances in diagnostic techniques continue to be realized for the evaluation of the patient with red cell alloimmunization in pregnancy. Noninvasive assessment of the fetal blood type through the use of free fetal DNA in maternal plasma in conjunction with middle cerebral artery Doppler
177 ultrasound for the detection of fetal anemia will likely make amniocentesis obsolete in the near future. Although currently intrauterine transfusion remains the mainstay of fetal treatment once anemia has been detected, selective manipulation of the maternal immune system will probably replace this technique in the coming years.
References 1. 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 192:171-177, 2005 2. van der Schoot CE, Tax GH, Rijnders RJ, de Haas M, Christiaens GC: Prenatal typing of Rh and Kell blood group system antigens: The edge of a watershed. Transfus Med Rev 17:31-44, 2003 3. Martin JA, Hamilton BE, Sutton PD, Ventura SJ, Menacker F, Munson ML: Births: Final data for 2002. Natl Vital Stat Rep 52:1-116, 2002 4. Cherif-Zahar B, Mattei 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 86: 398-400, 1991 5. Le Van Kim C, Cherif-Zahar B, Raynal V, Mouro I, Lopez M, Cartron JP, et al: Multiple Rh messenger RNA isoforms are produced by alternative splicing. Blood 80:1074-1078, 1992 6. 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 with the Rh D-negative blood group phenotype. Blood 95:12-18, 2000 7. Novaretti MC, Jens E, Pagliarini T, Bonifacio SL, Dorlhiac-Llacer PE, Chamone DA: Comparison of conventional tube test with diamed gel microcolumn assay for anti-D titration. Clin Lab Haematol 25:311-315, 2003 8. Bowman JM, Pollock JM, Manning FA, Harman CR, Menticoglou S: Maternal Kell blood group alloimmunization. Obstet Gynecol 79:239244, 1992 9. Kanter MH: Derivation of new mathematic formulas for determining whether a D- positive father is heterozygous or homozygous for the D antigen. Am J Obstet Gynecol 166:61-63, 1992 10. Chiu RW, Murphy MF, Fidler C, Zee BC, Wainscoat JS, Lo YM: Determination of RhD zygosity: Comparison of a double amplification refractory mutation system approach and a multiplex real-time quantitative PCR approach. Clin Chem 47:667-672, 2001 11. Moise KJ, Jr, Carpenter RJ: Jr: Chorionic villus sampling for Rh typing: Clinical implications. Am J Obstet Gynecol 168:1002-1003, 1993 (letter) 12. Bennett PR, Le Van Kim C, Colin Y, Warwick RM, Cherif-Zahar B, Fisk NM, et al: Prenatal determination of fetal RhD type by DNA amplification. N Engl J Med 329:607-610, 1993 13. Van den Veyver IB, Moise KJ Jr: Fetal RhD typing by polymerase chain reaction in pregnancies complicated by rhesus alloimmunization. Obstet Gynecol 88:1061-1067, 1996 14. Simsek S, Faas BH, Bleeker PM, Overbeeke MA, Cuijpers HT, van der Schoot CE, et al: Rapid Rh D genotyping by polymerase chain reactionbased amplification of DNA. Blood 85:2975-2980, 1995 15. Hopkins DF: Maternal anti-Rh(D) and the D-negative fetus. Am J Obstet Gynecol 108:268-271, 1970 16. Yankowitz J, Li S, Weiner CP: Polymerase chain reaction determination of RhC, Rhc, and RhE blood types: An evaluation of accuracy and clinical utility. Am J Obstet Gynecol 176:1107-1111, 1997 17. Spence WC, Maddalena A, Demers DB, Bick DP: Prenatal determination of genotypes Kell and Cellano in at-risk pregnancies. J Reprod Med 42:353-357, 1997 18. Hessner MJ, Pircon RA, Johnson ST, Luhm RA: Prenatal genotyping of Jk(a) and Jk(b) of the human Kidd blood group system by allele-specific polymerase chain reaction. Prenat Diagn 18:1225-1231, 1998 19. Hessner MJ, Pircon RA, Johnson ST, Luhm RA: Prenatal genotyping of
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the Duffy blood group system by allele-specific polymerase chain reaction. Prenat Diagn 19:41-45, 1999 Daniels G, Finning K, Martin P, Soothill P: Fetal blood group genotyping from DNA from maternal plasma: an important advance in the management and prevention of haemolytic disease of the fetus and newborn. Vox Sang 87:225-232, 2004 Lo YM, Zhang J, Leung TN, Lau TK, Chang AM, Hjelm NM: Rapid clearance of fetal DNA from maternal plasma. Am J Hum Genet 64:218224, 1999 Rouillac-Le Sciellour C, Puillandre P, Gillot R, Baulard C, Metral S, Le Van Kim C, et al: Large-scale pre-diagnosis study of fetal RHD genotyping by PCR on plasma DNA from RhD-negative pregnant women. Mol Diagn 8:23-31, 2004 Bevis DC: Blood pigments in haemolytic disease of newborn. J Obstet Gynaecol Br Emp 63:68-75, 1956 Liley AW: Liquor amnii analysis in the management of pregnancy complicated by rhesus sensitization. Am J Obstet Gynecol 82:1359-1370, 1961 Nicolaides KH, Rodeck CH, Mibashan RS, Kemp JR: Have Liley charts outlived their usefulness? Am J Obstet Gynecol 155:90-94, 1986 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’ gestation: A proposal for clinical management. Am J Obstet Gynecol 168:1370-1376, 1993 Hackney DN, Knudtson EJ, Rossi KQ, Krugh D, O’Shaughnessy RW: Management of pregnancies complicated by anti-c isoimmunization. Obstet Gynecol 103:24-30, 2004 Hughes L, Rossi K, Krugh D, O’Shaughnessy R: Management of pregnancies complicated by anti-Fya alloimmunization. Am J Obstet Gynecol 191:S164, 2004 Vaughan JI, Manning M, Warwick RM, Letsky EA, Murray NA, Roberts IA: Inhibition of erythroid progenitor cells by anti-Kell antibodies in fetal alloimmune anemia. N Engl J Med 338:798-803, 1998 Weiner CP, Widness JA: Decreased fetal erythropoiesis and hemolysis in Kell hemolytic anemia. Am J Obstet Gynecol 174:547-551, 1996 Weiner CP, Williamson RA, Wenstrom KD, Sipes SL, Grant SS, Widness JA: Management of fetal hemolytic disease by cordocentesis. I. Prediction of fetal anemia. Am J Obstet Gynecol 165:546-553, 1991 Weiner CP, Williamson RA, Wenstrom KD, Sipes SL, Widness JA, Grant SS, et al: Management of fetal hemolytic disease by cordocentesis. II. Outcome of treatment. Am J Obstet Gynecol 165:1302-1307, 1991 Weiner CP, Wenstrom KD, Sipes SL, Williamson RA: Risk factors for cordocentesis and fetal intravascular transfusion. Am J Obstet Gynecol 165:1020-1025, 1991 Nicolaides KH, Soothill PW, Clewell WH, Rodeck CH, Mibashan RS, Campbell S: Fetal haemoglobin measurement in the assessment of red cell isoimmunisation. Lancet 1:1073-1075, 1988 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 185:668673, 2001 Roberts AB, Mitchell JM, Lake Y, Pattison NS: Ultrasonographic surveillance in red blood cell alloimmunization. Am J Obstet Gynecol 184:1251-1255, 2001 Bahado-Singh R, Oz U, Mari G, Jones D, Paidas M, Onderoglu L: Fetal splenic size in anemia due to Rh-alloimmunization. Obstet Gynecol 92:828-832, 1998 Copel JA, Grannum PA, Green JJ, Belanger K, Hanna N, Jaffe CC, et al: Fetal cardiac output in the isoimmunized pregnancy: A pulsed Dop-
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pler-echocardiographic study of patients undergoing intravascular intrauterine transfusion. Am J Obstet Gynecol 161:361-365, 1989 Mari G: Noninvasive diagnosis by Doppler ultrasonography of fetal anemia due to maternal red-cell alloimmunization. N Engl J Med 342: 9-14, 2000 Opekes D, Seward G, Vandenbussche F, Kingdon J, Windrim R, Beyene J, et al: Minimally invasive management of rh alloimmunization: Can amniotic fluid delta OD450 be replaced by Doppler studies? A prospective study multicenter trial. Am J Obstet Gynecol 191:S3, 2004 Trevett T, Dorman K, Lamvu G, Moise KJ: Does antenatal maternal administration of phenobarbital prevent exchange transfusion in neonates with alloimmune hemolytic disease? Am J Obstet Gynecol 192: 478-482, 2005 Liley AW: Intrauterine transfusion of foetus in haemolytic disease. BMJ 2:1107-1109, 1963 Moise KJ Jr, Carpenter RJ Jr, Kirshon B, Deter RL, Sala JD, Cano LE: Comparison of four types of intrauterine transfusion: Effect on fetal hematocrit. Fetal Ther 4:126-137, 1989 Giannina G, Moise KJ Jr, Dorman K: A simple method to estimate the volume for fetal intravascular transfusion. Fetal Diagn Ther 13:94-97, 1998 Moise KJ Jr, Mari G, Fisher DJ, Huhta JC, Cano LE, Carpenter RJ Jr: Acute fetal hemodynamic alterations after intrauterine transfusion for treatment of severe red blood cell alloimmunization. Am J Obstet Gynecol 163:776-784, 1990 Radunovic N, Lockwood CJ, Alvarez M, Plecas D, Chitkara U, Berkowitz RL: The severely anemic and hydropic isoimmune fetus: changes in fetal hematocrit associated with intrauterine death. Obstet Gynecol 79:390-393, 1992 Schumacher B, Moise KJ Jr: Fetal transfusion for red blood cell alloimmunization in pregnancy. Obstet Gynecol 88:137-150, 1996 Koenig JM, Ashton RD, De Vore GR, Christensen RD: Late hyporegenerative anemia in Rh hemolytic disease. J Pediatr 115:315-318, 1989 Janssens HM, de Haan MJ, van Kamp IL, Brand R, Kanhai HH, Veen S: Outcome for children treated with fetal intravascular transfusions because of severe blood group antagonism. J Pediatr 131:373-380, 1997 Hudon L, Moise KJ Jr, 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 179:858-863, 1998 Moise KJ, Whitecar PW: Antenatal therapy for haemolytic disease of the fetus and newborn, in Hadley A, Soothill P (eds): Alloimmune Disorders in Pregnancy. Anaemia, Thrombocytopenia and Neutropenia in the Fetus and Newborn. Cambridge, UK, Cambridge University Press, 2002, pp 173-202 Voto LS, Mathet ER, Zapaterio JL, Orti J, Lede RL, Margulies M: Highdose gammaglobulin (IVIG) followed by intrauterine transfusions (IUTs): A new alternative for the treatment of severe fetal hemolytic disease. J Perinat Med 25:85-88, 1997 Moise KJ Jr: Management of rhesus alloimmunization in pregnancy. Obstet Gynecol 100:600-611, 2002 Seeho SK, Burton G, Leigh D, Marshall JT, Persson JW, Morris JM: The role of preimplantation genetic diagnosis in the management of severe rhesus alloimmunization: First unaffected pregnancy: Case report. Hum Reprod 20:697-701, 2005 Whitecar P, Farb R, Subramany L, Dorman K, Bagchee R, Moise K: Paternal leukocyte alloimmunization as a treatment for hemolytic disease of the newborn/fetus in the rabbit model. Am J Obstet Gynecol 185:S235, 2001 Stott LM, Barker RN, Urbaniak SJ: Identification of alloreactive T-cell epitopes on the Rhesus D protein. Blood 96:4011-4019, 2000
H
istorically, the fetal effects of maternal red blood cell (RBC) alloimmunization were undetectable until after the birth of an affected infant. These neonatal sequelae of maternal RBC alloimmunization became known as hemolytic disease of the newborn (HDN) or erythroblastosis fetalis. As a consequence of the declining rate of RhD alloimmunization secondary to the success of Rhesus immune globulin (RhIG), patients with more severe disease should be referred to a maternal-fetal medicine physician (formerly known as a perinatologist). These subspecialty-boarded individuals have completed a 2- or 3-year fellowship training program in highrisk obstetrics after a standard 4-year residency training period in obstetrics and gynecology. In the skilled hands of the maternal-fetal medicine physician, obstetrical ultrasound and direct access to the fetal circulation by cordocentesis has made the detection of fetal anemia a reality. Therefore it is now appropriate to refer to the perinatal effects of maternal RBC alloimmunization as hemolytic disease of the fetus and newborn (HDFN).
Significance and Incidence of RBC Antibodies More than 50 different RBC antigens can cause HDFN (Table 1). However, only anti-Rh(D), -Rh(c), and -Kell (K1) commonly appear in large case series of severe HDFN treated with intrauterine transfusion (IUT). In a recently published report of 254 fetuses referred to a tertiary care center for IUT in the Netherlands, 85% involved anti-D, 10% anti-K1, and 3.5% anti-c; there was one case each of anti-E, anti-e, and anti-Fya.1 A recent series of 1,133 Dutch women screened positive for antibodies associated with HDFN at 12 weeks of gestation2: anti-E was the most common antibody detected (23%) followed by anti-K (18.8%); anti-D was the third most common antibody detected (18.7%) followed by anti-c (10.4%), anti-C (7.5%), anti-Fya (5.8%), anti-Cw (5.7%), anti-S (3.5%), anti-Jka (2.5%), and miscellaneous other antibodies (3.7%). Current complete birth certificate data analyzed at the Centers for Disease Control found that in the year 2002, approximately 6.7 cases of Rhesus alloimmunization occurred per 1,000 live births in the United States.3
Genetics of the Rhesus Locus Division of Maternal-Fetal Medicine, University of North Carolina School of Medicine, Chapel Hill, NC. Address correspondence to Kenneth J. Moise Jr, MD, Director, Division of Maternal-Fetal Medicine, University of North Carolina School of Medicine, 214 MacNider Bldg CB#7516, Chapel Hill, NC 27599-7516. E-mail: [email protected]
0037-1963/05/$-see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1053/j.seminhematol.2005.04.007
Although three genes originally were proposed to encode for the Rh blood groups, the Rh locus is now known to consist of two genes located on the short arm of chromosome 1.4 Each is 10 exons in length and there is 96% homology between the genes. Production of two distinct proteins from the RhCE 169
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170 Table 1 Non-Rh(D) Antibodies and Associated HDFN Antigen System
Specific Antigen
Antigen System
Specific Antigen
Antigen System
Specific Antigen
Frequently associated with severe disease Kell Rh
-K (K1) -c
Colton
-Coa -Co3 -ELO -Dia -Dib -Wra -Wrb -Fya -Jsb -k (K2) -Kpa -Kpb -K11 -K22 -Ku -U1a -Jka -Ena -Far -Hil -Hut -M -Mia -Mta -MUT
Diego
Duffy Kell
Kidd MNS
Duffy Gerbich
Kell
-Fyb -Fy3 -Ge2 -Ge3 -Ge4 -Lsa -Jsa
Infrequently associated with severe disease MNS -Mur -MV -s -sD -S -U -Vw Rh -Bea -C -Ce -CW -ce -E -EW -Evans -G -Goa -Hr -Hro -JAL -Rh32 -Rh42 -Rh46 -STEM -Tar Associated with mild disease Kidd -Jkb -Jk3 MNS -Mit Rh -CX -DW -e -HOFM -LOCR
Scianna Other Ag’s
Rh Other
-Sc2 -Rd -Bi -Good -Heibel -HJK -Hta -Jones -Joslin -Kg -Kuhn -Lia -MAM -Niemetz -REIT -Reiter -Rd -Sharp -Vel -Zd
-Riv -RH29 -Ata -JFV -Jra -Lan
Reproduced with permission from Moise KJ: Hemolytic disease of the fetus and newborn, in Creasy RK, Resnik R, Iams J (eds): Maternal-Fetal Medicine, Principles and Practice (ed 5). Philadelphia, PA, Elsevier, Copyright © 2004.
gene probably occurs as a result of alternative splicing of messenger RNA.5 Further study of the RHD gene has revealed significant heterogeneity. Several of these genetic modifications result in a lack of expression of the Rh(D) phenotype. One such example is the RHD pseudogene, which has been found in 69% of South African blacks and 24% of African-Americans.6 All 10 exons of the RHD gene are present, but transcription of the gene into a messenger RNA product does not occur due to the presence of a stop codon in the intron between exons 3 and 4. Similarly, the Ccdes gene has been detected in 22% of African-Americans; it appears to contain exons 1, 2, 9, and 10 as well as a portion of exon 3 of the original RH(D) gene, with other exons being duplicated from the RhCE gene. The presence of these genes in an Rh(D)-negative pregnant patient has
important implications for the prenatal diagnosis of the fetal blood type (see below).
Diagnostic Methods Maternal Antibody Determination Once a maternal antibody screen reveals the presence of anti-D, an antiglobulin titer is the first step in the evaluation of the RhD-sensitized patient during her first affected pregnancy. Sera from each titer should be frozen and used for determining subsequent values, in tandem for purposes of comparison. Many patients and obstetricians will not realize that a one tube change in dilution does not represent a true increase or decline in the level of antibody. Titers are usually
Red blood cell alloimmunization in pregnancy
171
Table 2 Incidence of Paternal Heterozygosity (%) Based on Serology, Ethnic Background, and Number of Previous RhD-Positive Offspring No. of RhDⴙ Infants Caucasian
DCce DCe DcEe DcE DCcEe Dce
Black
Hispanic
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
90 9 90 13 11 94
82 5 82 7 6 89
69 2 69 4 3 80
53 1 53 2 2 66
36 0.6 36 0.9 0.8 50
22 0.3 22 0.5 0.4 33
41 19 37 1 10 54
26 11 23 0.5 5 37
15 6 13 0.3 3 23
8 3 7 0.1 1 13
4 1 4 0.1 0.7 7
2 0.7 2 0 0.3 4
85 5 85 2 12 92
74 2 74 0.9 6 85
59 1 59 0.5 3 74
42 0.6 42 0.2 2 59
26 0.3 26 0.1 0.8 42
15 0.1 15 0.1 0.4 26
Reproduced with permission from Moise KJ: Modern management of Rhesus alloimmunzation in pregnancy. Obstet Gynecol 100:600 – 611, 2002. Copyright 2002, Lippincott Williams & Wilkins.
repeated at 4-week intervals until the middle of the second trimester (24 weeks’ gestation); thereafter, they are repeated every 2 weeks. A critical titer is defined as the antibody titer associated with a significant risk for hydrops fetalis; when present, further fetal surveillance is warranted. This value will vary with institution and methodologies, but in most centers a critical titer for anti-D of 32 is usually used in the first affected pregnancy. Standard tube methodology should be utilized in determining the critical titer, as newer gel microcolumn assays have been reported to result in titers that are 3.4-fold higher.7 Maternal titers are not useful in clinical management after the first affected pregnancy. The generally recommended critical titer for other irregular RBC antibodies is 32. The notable exceptions are the two antibodies to antigens in the Kell system, anti-K and anti-k, for which a lower critical value of 8 has been suggested for maternal antibody.8
Fetal Blood Typing The initial step in determining the fetal blood type is to establish paternal zygosity. Since the RHC/c and E/e genes are inherited in a closely linked fashion to RHD, antisera for these antigens can be used with gene frequency tables. Ethnicity must be incorporated in to the calculation of the likelihood of heterozygosity, as very disparate results can be obtained with similar serologic findings. In addition, Bayesian analysis can be employed to modify the incidence of heterozyosity based on the paternal history of previous Rh(D)-positive offspring (Table 2).9 When the lines of ethnicity are murky, the use of serology and historic population studies become less reliable means of determining paternal zygosity. Quantitative polymerase chain reaction (PCR) has been used with some accuracy to compare the number of copies of the Rh(D) gene in a particular individual to a baseline control gene such as human albumin.10 In addition, it is now recognized that, in the majority of individuals with a gene deletion as the etiology of their Rh(D)-negative status, there are two silent gene segments flanking the main RHD gene. The loss of the RHD gene results in a combined hybrid of these two Rhesus boxes, which can be detected through restriction fragment length polymorphism analysis.2 Although not currently commer-
cially available, this assay will probably be used in the near future to determine paternal zygosity for RHD. After paternal testing has revealed the possibility of a heterozygous state for RHD, fetal testing is indicated. Chorion villus biopsy (placental aspiration under ultrasound guidance) can be undertaken at 10 to 14 weeks’ gestation to obtain genetic material for detection of the RHD gene. The major disadvantage of this method is that disruption of the chorion villi during the procedure can result in fetomaternal hemorrhage and a subsequent rise in maternal titer thereby worsening the fetal disease.11 This procedure should be discouraged unless the patient plans to electively abort an antigen-positive fetus that is detected. In 1990, amniocentesis was described as a reliable method for assessing the fetal blood type through DNA testing.12 Extensive experience using amniocentesis to determine fetal blood type has revealed rare discrepancies in fetal RHD typing. A false negative result occurs when amniotic fluid analysis finds an RHD-negative fetus that is confirmed to be RHDpositive by serology after birth. In this case, the usual fetal surveillance techniques would not be employed and there would be the potential for perinatal loss. In one series of 500 amniocenteses, false negative results occurred in 1.5% of cases.13 The most likely etiology for discordance is either erroneous paternity or a rearrangement at the paternal RHD gene locus. Such gene rearrangements occur in approximately 2% of individuals14 and thus most laboratories offering fetal RBC typing on amniotic fluid require an accompanying paternal blood sample. Verifying that paternal serology and DNA testing reveal equivalent results confirms that a paternal gene rearrangement is not a potential source for error in fetal testing. Alternatively, the use of multiplex PCR targeting at least two different exons of the RHD gene can decrease the likelihood of nondetection of a gene rearrangement. In situations where paternity is not assured or there is no partner available to contribute a blood sample for confirmation of PCR primers, the result of a RHD-negative fetal blood type from amniotic fluid DNA analysis must be considered suspect. Serial maternal titers in patients with Rh(D) alloimmunization who subsequently delivered Rh(D)-negative off-
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Figure 1 Algorithm for clinical management of a patient with red cell sensitization in the first affected pregnancy. (Modified from Moise, KJ: Rh disease: It’s still a threat. Contemp Obstet Gynecol 49:34-48, 2004.)
spring were noted to rise by fourfold in less than 2% of cases.15 In situations of questionable paternity or lack of availability of a paternal blood sample, a repeat maternal antiglobulin titer obtained at 4 to 6 weeks after the results of the amniocentesis offers confirmation (Fig 1); if a fourfold or greater rise in maternal antibody titer is noted (32 to 128), then an Rh(D)-negative result on amniotic fluid should be questioned. Repeat amniocentesis to evaluate the delta optical density at 450 nm (⌬OD450) or fetal blood sampling to determine the fetal Rh(D) status using serologic techniques may be advisable. If the maternal race is black, then the presence of a maternal pseudogene or Ccdes gene should be considered in the scheme of fetal testing. The presence of one of these genes in the fetus can lead to a false positive diagnosis; the amniotic fluid result would be Rh(D)-positive yet the fetus would be found to be Rh(D)-negative by serology after birth, leading to unnecessary fetal interventions with their inherent risks. For this reason, a maternal blood sample should always accom-
pany the amniotic fluid aliquot sent for fetal RHD testing in an effort to rule out the presence of a maternal RhD pseudogene or Ccdes gene. If the maternal sample is positive for one of these variants, then fetal testing for the gene should also be undertaken. DNA analysis of amniotic fluid for fetal typing has been successfully employed for most major RBC antigens associated with HDFN including Rh(D), Rh(C/c), Rh(E/e), K/k, Jka/Jkb, and Fya/Fyb.12,16-19 Fetal Rh(D) determination through noninvasive testing is now routine in other countries.20 Free fetal DNA is cleared from the maternal plasma within minutes after delivery, thereby eliminating the possibility of contamination from a previous gestation.21 In a series of 893 Rh(D)-negative pregnant patients, 42 samples were noted to exhibit a nonfunctional or rearranged RhD gene. After these were excluded, free DNA was accurate in 99.5% of cases in determining the fetal RhD type.22 Once the issue of an aberrant RH(D) gene has been addressed, RHD-positive results on free DNA can be
Red blood cell alloimmunization in pregnancy considered reliable as RHD-positive genetic material cannot be from a maternal source. A fetal RHD-negative result is more problematic. If fetal DNA fails to amplify in a background of overwhelming maternal DNA in plasma, an RHDnegative result will be obtained. One internal control is the detection of the SRY gene found in association with male fetuses; the presence of this gene in free DNA indicates that fetal DNA is present and an Rh(D)-negative result is reliable.20 For a female fetus, DNA polymorphisms noted in the maternal white blood cells can be used as an internal control.20 If different polymorphisms than those found in the mother are present in the plasma sample, fetal DNA is present. Again, in this situation the finding of an Rh(D)negative fetus can be considered reliable. In as many as 4% of cases, DNA polymorphisms are not informative so the RhDnegative fetal result remains questionable; a repeat maternal sample could be submitted or amniocentesis could be undertaken to determine the fetal RhD type. Only free fetal DNA testing for RHD typing of the fetus is currently performed, although assays for K, RHE, and RHc will be available in the near future.2
Amniocentesis for ⌬OD450 Since first introduced to clinical practice by Bevis,23 spectral analysis of amniotic fluid at 450 nm (⌬OD450) has been used to measure the level of bilirubin, an indirect indicator of the degree of fetal hemolysis. Liley24 proposed a management scheme involving three zones based on gestational ages between 27 and 42 weeks. Zone 1 indicated mild HDFN while zone 3 was associated with fetal death within 10 days. Extrapolated Liley curves to gestational ages earlier than 27 weeks have proven erroneous in diagnosing fetal anemia.25 A modified ⌬OD450 curve for such situations has been published by Queenan et al26: four zones were proposed, with the upper two zones being termed the Rh-positive, affected zone and the intrauterine death zone. Serial amniocenteses have been successfully employed in the past to follow alloimmunized pregnancies to non-Rh(D) antibodies.27,28 The notable exception has been in patients sensitized to Kell antigens. In vitro and in vivo studies have shown that fetal anemia secondary to anti-K is secondary to two mechanisms: hemolysis and erythropoietic suppression.29,30 Amniotic fluid ⌬OD450 values do not reflect the degree of fetal anemia as accurately as in cases involving anti-RhD. As a result some recommend a lower threshold of the 65 percentile of zone 2 of the Liley curve to proceed to cordocentesis.8 Alternatively, middle cerebral artery (MCA) Doppler measurements are probably more accurate than ⌬OD450 values in these cases (see below). 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. All attempts should be made to avoid transplacental passage of the needle, since this can lead to fetomaternal hemorrhage and a rise in maternal antibody titer. A rising or plateauing ⌬OD450 value that reaches the eightieth percentile of zone 2 of the Liley curve or a value that enters the upper
173 portion of the Rh-positive, affected zone of the Queenan curve requires investigation by fetal blood sampling.
Fetal Blood Sampling Ultrasound-directed fetal blood sampling (FBS; also cordocentesis, percutaneous umbilical blood sampling, and funipuncture) allows direct assess to the fetal circulation to obtain important laboratory values such as fetal blood type, hematocrit, direct Coombs, reticulocyte count, and total bilirubin. In this procedure, a 20- or 22-gauge needle is directed into the amniotic cavity under continuous ultrasound guidance. The umbilical cord is targeted at its insertion into the placenta. In the case of an anterior placenta, the needled is directed through the placental mass into the cord root. Although a free-floating cord can be used, the ease of mobility in the liquid environment makes for a more difficult procedure. Procedure-related complications include anamnestic response in maternal antibody titer, fetal exsanguination from the puncture site, transient fetal bradycardia, cord hematoma, and fetal death.31,32 One large series reported a perinatal loss rate of 1% after cordocentesis for diagnostic purposes.33 Although there are three potential vessels in the umbilical cord for vascular access—two arteries and one vein—the umbilical vein represents a larger target and has been associated with a lower incidence of fetal bradycardia as compared to puncture of an umbilical artery.1 For these reasons, cordocentesis is reserved for patients with elevated ⌬OD450 values or elevated peak MCA Doppler velocities (see below).
Ultrasound Perhaps the greatest advance in the management of the alloimmunized pregnancy has been the use of ultrasound. Gestational age can be accurately established in order to evaluate fetal parameters that vary with fetal age, including hemoglobin, ⌬OD450 levels, and peak MCA Doppler velocities. Hydrops fetalis is defined as the presence of extracellular fluid in at least two fetal compartments. Often ascites is the first sign of impending hydrops, with scalp edema and pleural effusions noted with worsening anemia. When hydrops is present, fetal hemoglobin deficits of 7 to 10 g/dL from the mean hemoglobin value for the corresponding gestational age can be expected34— unfortunately, this represents the end-stage state of fetal anemia. Survival with intrauterine transfusion is markedly reduced in these cases, and therefore many investigators have sought alternative ultrasound parameters that could predict the early onset of anemia.35 Although assessment of fetal hepatosplenomegaly using ultrasound has demonstrated some ability to predict fetal anemia, Doppler ultrasound studies of the peak velocity in the MCA have been most predictive (Fig 2).36,37 The severely anemic fetus exhibits an increased cardiac output in an effort to enhance oxygen delivery to peripheral tissues.38 In addition, fetal anemia is associated with a lower blood viscosity, which produces less shearing forces in blood vessels, resulting in increased blood velocities. In a recent study 111 fetuses at risk for fetal anemia secondary to HDFN were assessed using
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Figure 2 Middle cerebral artery Doppler determination; arrows denote anterior and posterior fetal MCA vessels. Dotted line and box denoted angle of insonation and Doppler gate for measurement of the peak velocity.
receiver operator curve analysis.39 A threshold value of 1.5 multiples of the median (MoMs) for the peak MCA velocity was used to predict moderate to severe anemia (⬍0.65 MoMs for fetal hemoglobin). This degree of anemia was predicted with a sensitivity of 100% and a false positive rate of 10%. A recent prospective, multicenter trial confirmed these initial results with a sensitivity of 88% and a negative predictive rate of 89%.40 Many centers have now replaced serial amniocenteses for ⌬OD450 with serial MCA Doppler. MCA measurements can be obtained reliability as early as 18 weeks’ gestation. Studies are repeated every 1 to 2 weeks depending on the trend (Fig 3); values can be plotted on standard curves or converted to MoMs using internet-based calculators, but values after 35 weeks’ gestation are associated with a higher rate of false positives. If MCA Doppler were routinely employed, more than 70% of invasive testing could be avoided using this modality to monitor alloimmunized pregnancies.
Clinical Management The approach using available diagnostic tools is based on the patient’s past history of fetal or neonatal manifestations. The patient’s first antigen-sensitized pregnancy usually involves
Figure 3 Serial MCA Doppler studies in one patient who required intrauterine transfusion. Vertical axis represents the MCA velocity in cm/s; the horizontal axis represents advancing gestational age in weeks. Zone A denotes mild anemia zone; Zone B denotes moderate to severe anemia zone. Cordo, cordocentesis; MoM, multiples of the median. (Reproduced with permission from Moise KJ: Hemolytic disease of the fetus and newborn, in Creasy RK, Resnik R, Iams J (eds): Maternal-Fetal Medicine, Principles and Practice (ed 5): Philadelphia, PA, Elsevier, Copyright © 2004).
Red blood cell alloimmunization in pregnancy clinically minimal HDFN; subsequent gestations are associated with a worsening degree of anemia.
First Affected Pregnancy Once sensitization to a significant RBC antigen is detected, maternal titers are repeated every month until approximately 24 weeks; titers are repeated every 2 weeks thereafter (Fig 1). Paternal blood is drawn to determine antigen status and zygosity through serologic testing. Once a critical maternal titer is reached (usually 32), serial MCA Doppler examinations are initiated at approximately 24 weeks’ gestation and then repeated every 1 to 2 weeks depending on their trend. In remote geographic areas where access to a referral center for MCA Doppler is impractical, serial amniocenteses for ⌬OD450 starting at 24 weeks and repeated at 10-day to 2-week intervals is acceptable. In cases of a heterozygous paternal phenotype, an amniotic fluid sample obtained at the time of the initial amniocentesis accompanied by a maternal and paternal blood sample should be sent to a DNA reference laboratory to determine the fetal antigen status. In the case of an antigen-negative paternal blood type (with assured paternity) or a fetal antigen-negative genotype on amniotic fluid analysis, no further maternal or fetal monitoring is needed. If there is evidence of an antigen-positive fetus (a homozygous paternal phenotype or antigen-positive fetus by PCR testing on amniotic fluid), serial fetal surveillance is indicated. If an MCA Doppler shows ⱖ1.5 MoMs, cordocentesis should be undertaken at an experienced referral center with blood readied for intrauterine transfusion if the fetal hematocrit is found to be less than 30%. Alternatively, if the ⌬OD450 rises into or plateaus in the upper portion of the Rh-positive, affected zone of the Queenan curve or the eightieth percentile of the Liley curve, cordocentesis should be planned. If the peak MCA value remains at less than 1.5 MoMs, amniocentesis at 35 weeks’ gestation should be considered. When fetal pulmonary maturity is present and the ⌬OD450 is not in the upper portion of the Rh-positive, affected zone of the Queenan curve, delivery should be induced in 2 weeks. This time interval enables fetal hepatic maturity to occur and limits the need for exchange transfusion secondary to neonatal hyperbilirubinemia. If fetal pulmonary immaturity is noted at the 35-week amniocentesis and the ⌬OD450 is in the Rh-positive, affected zone of the Queenan curve, administration of maternal phenobarbital (30 mg orally three times per day) with induction 1 week later decreases the probability of later neonatal exchange transfusion.41 Finally, if results indicate fetal lung immaturity with a ⌬OD450 that is not in the Rh-positive, affected zone of the Queenan curve, a repeat amniocentesis should be undertaken in 2 weeks. Management of a pregnancy with serial amniocenteses for ⌬OD450 would follow this same management plan in the late third trimester.
Previously Affected Fetus or Infant If there is a history of a previous perinatal loss related to HDFN, intrauterine transfusion, or neonatal exchange trans-
175 fusion, the patient should be referred to a tertiary care center experienced in the management of the severely alloimmunized pregnancy (Fig 4). In these cases, maternal titers are not predictive of the degree of fetal anemia. When the paternal phenotype is heterozygous, amniocentesis at 15 weeks’ gestation to determine the fetal antigen status is indicated. Serial MCA Doppler measurements should begin at 18 weeks’ gestation and be repeated every 1 to 2 weeks. Alternatively, serial amniocenteses for measurement of ⌬OD450 can be used with the Queenan curve for reference values beginning at 18 weeks. Subsequent surveillance is similar as for the first affected pregnancy.
Intrauterine Transfusion Since it was first introduced in 1963 by William Liley,42 IUT of RBCs has proven lifesaving for cases of severe HDFN. The original approach was to infuse donor RBCs into the peritoneal cavity of the fetus, where they would be subsequently absorbed through the diaphragmatic lymphatics. Today, IUTs are performed as direct infusions of RBCs into the umbilical cord or into the intrahepatic portion of the umbilical vein of the fetus. Some centers continue to use the intraperitoneal approach as part of a combined technique with intravascular transfusion in an effort to create a reservoir of RBCs between procedures.43 Typically a freshly donated cytomegalovirus-negative unit of type O, Rh(D)-negative RBCs is cross-matched to a maternal sample; the unit is leukoreduced, irradiated with 25 Gy to prevent graft-versus-host reaction, and then packed to a final hematocrit of approximately 75% to 80%. A unit with a hematocrit of less than 75% could cause volume overload in the fetus, while a hematocrit of greater than 85% is too viscous and difficult to transfuse through the long, small-diameter procedure needle. The initial step in the procedure is to introduce the needle under continuous ultrasound guidance into the umbilical vein. A sample of fetal blood is obtained for initial hematocrit. Optimally, the sample is processed as a spun hematocrit or through the use of an automated hemocytometer located in the operating room. A short-term paralytic agent such as vecuronium or atracurium is administered to the fetus to cause cessation of fetal movement. The amount of packed RBCs to be infused is based on the estimated fetal weight as determined by ultrasound and standardized formulas.44 A final fetal hematocrit of approximately 40% is targeted. Severely anemic fetuses in the early second trimester do not tolerate acute correction of their hematocrit to normal values,45 and the initial hematocrit should not be increased by more than four-fold at the time of the first procedure.46 A repeat intravascular transfusion is then performed within 48 hours to correct the fetal hematocrit into the normal range. At the conclusion of the procedure, a small aliquot of blood is obtained to measure the hematocrit as well as the percentage of fetal versus adult hemoglobin-containing RBCs through either a Kleihauer-Betke stain or fetal flow cytometry. After the first IUT, subsequent procedures can be empirically scheduled at 14-day intervals until suppression of
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Figure 4 Algorithm for clinical management of a patient with red cell sensitization and a previously affected fetus or infant. (Modified from Moise KJ: Rh disease: It’s still a threat. Contemp Obstet Gynecol 49:34-48, 2004.)
fetal erythropoiesis is observed, usually by the third IUT. Thereafter, the interval for repeat procedures is based on the decline in hematocrit for the individual fetus, usually every 3 to 4 weeks. A final procedure is usually not performed past 35 weeks’ gestation and the patient is scheduled for delivery approximately 3 weeks later.
Outcome Survival after IUT varies by center, experience, and the presence of hydrops fetalis. Overall survival in one review was 84%; up to one fourth fewer hydropic fetuses survive with IUT (70%) as compared to fetuses who undergo their first IUT when they are not hydropic (92%).47 The experience of a single treatment center with 254 fetuses receiving 740 IUTs indicated an overall survival rate of 89%.1 The practice of prolonging the gestation of the treated fetus with HDFN until near term has resulted in a rare necessity for
a neonatal exchange transfusion. Typically, these infants are born with a virtual absence of reticulocytes and a RBC population consisting mainly of transfused blood. The blood bank may be confused if cord blood at delivery is submitted for neonatal RBC typing—the neonate will be typed as O, Rh(D)-negative reflecting the antigen status of the donor blood used for the IUTs! Elevated levels of circulating maternal antibodies in the neonatal circulation in conjunction with suppression of the fetal bone marrow will result in the need for neonatal RBC top-up transfusions at approximately 1 month of age in 50% of these infants.48 These children should be monitored weekly by hematocrits and reticulocyte counts until there is evidence of recovery of hematopoietic function. Only limited data are available for counseling the patient regarding long-term outcome, since fetuses with severe anemia and hydrops are likely to survive today secondary to the use of intravascualr transfusions. Short-term studies at up to
Red blood cell alloimmunization in pregnancy 2 years of age have revealed that more than 90% of infants are neurologically normal despite the history of hydrops fetalis.49,50
Other Treatment Modalities Prior to the advent of the IUT, plasmapheresis represented one of the few therapeutic modalities for severe HDFN. The literature consists mainly of single patients or relatively small series. Despite these limitations, a review of the published cases reveals a perinatal survival rate of 69%.51 Intravenous immune globulin (IVIG) has been used effectively as the sole antenatal treatment for HDFN. In a case-control study of 69 patients, IVIG given prior to 20 weeks’ gestation followed by IUT was compared to the use of only IUTs after 20 weeks.52 Seventy-three percent of the IVIG group and 56% of the IUT group had experienced previous perinatal losses. Hydrops at the first IUT occurred in one fourth of the IVIG/IUT group as compared to three fourths of the IUT-only group. Importantly, the first IUT was performed a median of 1.5 weeks later in gestation in the patients that had received IVIG. Some experts have proposed a combined approach in patients with a previous perinatal loss in the early second trimester, when technical limitations make the success of IUT unlikely.53 Plasmapheresis is started at 12 weeks’ gestation and repeated three times in that week. The maternal titer should be expected to be reduced by 50%. IVIG is then given to replace the globulin fraction as a 2-g/kg loading dose after the third plasmapheresis; this is followed by 1 g/kg/wk of IVIG until 20 weeks’ gestation.
Future Therapeutic Options Patients with high antibody titers and recurrent perinatal loss in the second trimester have few options other than artificial insemination with RBC antigen-negative donor semen, surrogate pregnancy, or preimplantation diagnosis.54 Future therapy will probably involve selective manipulation of the maternal immune system. In vitro data and clinical case reports suggest that maternal alloantibodies to paternal leukocytes may result in an Fc blockade, thereby protecting the fetal RBCs from hemolysis in cases of Rh(D) alloimmunization. In a rabbit model for HDFN, alloimmunization to paternal leukocytes resulted in fetal hemoglobin levels that approached normal in does that had been previously sensitized to RBCs.55 As an alternative strategy, four peptides have been associated with the proliferation of T-helper cells involved in the development of antibody to the RhD antigen; therapeutic administration of these peptides might ameliorate an established anti-D response, preventing severe HDFN in a subsequent pregnancy.56
Conclusion Major advances in diagnostic techniques continue to be realized for the evaluation of the patient with red cell alloimmunization in pregnancy. Noninvasive assessment of the fetal blood type through the use of free fetal DNA in maternal plasma in conjunction with middle cerebral artery Doppler
177 ultrasound for the detection of fetal anemia will likely make amniocentesis obsolete in the near future. Although currently intrauterine transfusion remains the mainstay of fetal treatment once anemia has been detected, selective manipulation of the maternal immune system will probably replace this technique in the coming years.
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