RED CELL ALLOIMMUNIZATION
Fetal disorders may be acquired—such as alloimmunization, they may be genetic—congenital adrenal hyperplasia or α4-thalassemia, or they may be sporadic developmental abnormalities—like many structural malformations. Reviewed in this chapter are fetal anemia and thrombocytopenia, along with immune and nonimmune fetal hydrops. Hydrops is perhaps the quintessential fetal disorder, as it can be a manifestation of severe illness from a wide variety of etiologies. Fetal structural malformations are reviewed in Chapter 10; genetic abnormalities are reviewed in Chapters 13 and 14; and other conditions amenable to fetal medical and surgical treatment are reviewed in Chapter 16. Because congenital infections arise as a result of maternal infection or colonization, they are considered in Chapters 64 and 65.
Of the many causes of fetal anemia, the most common is red cell alloimmunization, which results from transplacental passage of maternal antibodies that destroy fetal red cells. Alloimmunization leads to overproduction of immature fetal and neonatal red cells—erythroblastosis fetalis–a condition now referred to as hemolytic disease of the fetus and newborn (HDFN). Several congenital infections are also associated with fetal anemia, particularly parvovirus B19, discussed in Chapter 64 (p. 1244). In Southeast Asian populations, α4-thalassemia is a common cause of severe anemia and nonimmune hydrops. Fetomaternal hemorrhage occasionally creates severe fetal anemia and is discussed on page 312. Rare causes of anemia include red cell production disorders—such as Blackfan-Diamond anemia and Fanconi anemia; red cell enzymopathies—glucose-6-phosphate dehydrogenase deficiency and pyruvate kinase deficiency; red cell structural abnormalities—hereditary spherocytosis and elliptocytosis; and myeloproliferative disorders—leukemias. Anemia may be identified through fetal blood sampling as described in Chapter 14 (p. 300) or by Doppler evaluation of the fetal middle cerebral artery (MCA) peak systolic velocity as described on page 310.
Progressive fetal anemia from any cause leads to heart failure, hydrops fetalis, and ultimately death. Fortunately, the prevention of Rh D alloimmunization with anti-D immune globulin, and the identification and treatment of fetal anemia with MCA Doppler studies and intrauterine transfusions, respectively, have dramatically changed the prevalence and course of this otherwise devastating disorder. Severely anemic fetuses transfused in utero have survival rates exceeding 90 percent, and even in cases of hydrops fetalis, survival rates approach 80 percent (Lindenburg, 2013; van Kamp, 2001).
Red Cell Alloimmunization
There are currently 30 different blood group systems and 328 red cell antigens recognized by the International Society of Blood Transfusion (Storry, 2011). Although some of these are immunologically and genetically important, many are so rare as to be of little clinical significance. Any individual who lacks a specific red cell antigen may produce an antibody when exposed to that antigen. Such antibodies can prove harmful to that individual if she receives an incompatible blood transfusion, and they may be harmful to her fetus during pregnancy. Accordingly, blood banks routinely screen for erythrocyte antigens. These antibodies may also be harmful to a mother’s fetus during pregnancy. As noted, maternal antibodies formed against fetal erythrocyte antigens may cross the placenta to cause fetal red cell lysis and anemia.
Typically, a fetus inherits at least one red cell antigen from the father that is lacking in the mother. Thus, the mother may become sensitized if enough fetal erythrocytes reach her circulation to elicit an immune response. Even so, alloimmunization is uncommon for the following reasons: (1) low prevalence of incompatible red cell antigens; (2) insufficient transplacental passage of fetal antigens or maternal antibodies; (3) maternal-fetal ABO incompatibility, which leads to rapid clearance of fetal erythrocytes before they elicit an immune response; (4) variable antigenicity; and (5) variable maternal immune response to the antigen.
In population-based screening studies, the prevalence of red cell alloimmunization in pregnancy is approximately 1 percent (Howard, 1998; Koelewijn, 2008). Most cases of severe fetal anemia requiring antenatal transfusion are attributable to anti-D, anti-Kell, or anti-c alloimmunization.
A blood type and antibody screen are routinely assessed at the first prenatal visit, and unbound antibodies in maternal serum are detected by the indirect Coombs test (Chap. 9, p. 174). With positive results, specific antibodies are identified, their immunoglobulin subtype is determined as either IgG or IgM, and the titer is quantified. Only IgG antibodies are of concern because IgM antibodies do not cross the placenta. Selected antibodies and their potential to cause fetal hemolytic anemia are listed in Table 15-1. The critical titer is the level at which significant fetal anemia could potentially develop. This may be different for each antibody, is determined individually by each laboratory, and usually ranges between 1:8 and 1:32. If the critical titer for anti-D antibodies is 1:16, a titer ≥ 1:16 indicates the possibility of severe hemolytic disease. An important exception is Kell sensitization, which is discussed on page 308.
TABLE 15-1. Minor Red Cell Antigens and Their Relationship to Fetal Hemolytic Disease
CDE (Rh) Blood Group Incompatibility
The rhesus system includes five red cell proteins or antigens: C, c, D, E, and e. No “d” antigen has been identified, and Rh D-negativity is defined as the absence of the D antigen. Although most people are Rh D positive or negative, more than 200 D antigen variants exist (Daniels, 2013).
CDE antigens are clinically important. Rh D-negative individuals may become sensitized after a single exposure to as little as 0.1 mL of fetal erythrocytes (Bowman, 1988). The two responsible genes—RHD and RHCE—are located on the short arm of chromosome 1 and are inherited together, independent of other blood group genes. Their incidence varies according to racial and ethnic origin. Nearly 85 percent of non-Hispanic white Americans are Rh D-positive, as are approximately 90 percent of Native Americans, 93 percent of African Americans and Hispanic Americans, and 99 percent of Asian individuals (Garratty, 2004).
The prevalence of Rh D alloimmunization complicating pregnancy ranges from 0.5 to 0.9 percent (Howard, 1998; Koelewijn, 2008; Martin, 2005). Without anti-D immune globulin prophylaxis, an Rh D-negative woman delivered of an Rh D-positive, ABO-compatible infant has a 16-percent likelihood of developing alloimmunization. Two percent will become sensitized by the time of delivery, 7 percent by 6 months postpartum, and the remaining 7 percent will be “sensibilized”—producing detectable antibodies only in a subsequent pregnancy (Bowman, 1985). If there is ABO incompatibility, the Rh D alloimmunization risk is approximately 2 percent without prophylaxis (Bowman, 2006). The reason for the differing rates relative to ABO blood type results from erythrocyte destruction of ABO-incompatible cells and thus limitation of sensitizing opportunities. Rh D sensitization also may occur following first-trimester pregnancy complications, prenatal diagnostic procedures, and maternal trauma (Table 15-2).
TABLE 15-2. Causes of Fetomaternal Hemorrhage Associated with Red Cell Antigen Alloimmunizationa
Fetal death (any trimester)
Chorionic villus sampling
Fetal blood sampling
Unexplained vaginal bleeding during pregnancy
External cephalic version
aFor each of the above, anti-D immune globulin is recommended.
Data from The American Academy of Pediatrics and American College of Obstetricians and Gynecologists, 2012.
The Rh C, c, E, and e antigens have lower immunogenicity than the Rh D antigen, but they too can cause hemolytic disease. Sensitization to E, c, and C antigens complicates approximately 0.3 percent of pregnancies in screening studies and accounts for about 30 percent of red cell alloimmunization cases (Howard, 1998; Koelewijn, 2008). Anti-E alloimmunization is the most common, but the need for fetal or neonatal transfusions is significantly greater with anti-c alloimmunization than with anti-E or anti-C (Hackney, 2004; Koelewijn, 2008).
The Grandmother Effect. In virtually all pregnancies, small amounts of maternal blood enter the fetal circulation. Real-time polymerase chain reaction (PCR) has been used to identify maternal Rh D-positive DNA in peripheral blood from preterm and full-term Rh D-negative newborns (Lazar, 2006). Thus, it is possible for an Rh D-negative female fetus exposed to maternal Rh D-positive red cells to develop sensitization. When such an individual reaches adulthood, she may produce anti-D antibodies even before or early in her first pregnancy. This mechanism is called the grandmother theory because the fetus in the current pregnancy is jeopardized by maternal antibodies that were initially provoked by his or her grandmother’s erythrocytes.
Alloimmunization to Minor Antigens
Because routine administration of anti-D immunoglobulin prevents anti-D alloimmunization, proportionately more cases of hemolytic disease are caused by red cell antigens other than D—also known as minor antigens (see Table 15-1). Kell antibodies are among the most frequent. Duffy group A antibodies—anti-Fya—are also fairly common, as are anti-MNSs and anti-Jka—Kidd group (Geifman-Holtzman, 1997). Most cases of sensitization to minor antigens result from incompatible blood transfusions (American College of Obstetricians and Gynecologists, 2012). If an IgG red cell antibody is detected and there is any doubt as to its significance, the clinician should err on the side of caution, and the pregnancy should be evaluated for hemolytic disease.
There are only a few blood group antigens that pose no fetal risk. Lewis antibodies—Lea and Leb, as well as I antibodies, are cold agglutinins. They are predominantly IgM and are not expressed on fetal red cells. Another antibody that does not cause fetal hemolysis is Duffy group B—Fyb.
Kell Alloimmunization. Approximately 90 percent of whites and up to 98 percent of African Americans are Kell negative. Kell type is not routinely determined, and approximately 90 percent of Kell sensitization cases result from transfusion with Kell-positive blood. Thus, transfusion history is important.
If Kell sensitization develops from maternal–fetal incompatibility, it may develop more rapidly and may be more severe than with sensitization to Rh D and other blood group antigens. This is because Kell antibodies attach to fetal bone marrow erythrocyte precursors and prevent a hemopoietic response to anemia. With fewer erythrocytes produced, there is less hemolysis. Because of these vicissitudes, severe anemia may not be predicted by either the maternal Kell antibody titer or the amnionic fluid bilirubin level (p. 310). One option is to use a lower critical titer—1:8—for Kell sensitization (Moise, 2012). The American College of Obstetricians and Gynecologists (2012) has recommended that antibody titers not be used to monitor Kell-sensitized pregnancies. Van Wamelen and colleagues (2007) have advocated MCA Doppler studies beginning at 16 to 17 weeks’ gestation for pregnancies with anti-Kell titers ≥ 1:2.
ABO Blood Group Incompatibility
Incompatibility for the major blood group antigens A and B is the most common cause of hemolytic disease in newborn infants, but it does not cause appreciable hemolysis in the fetus. Approximately 20 percent of newborns have ABO blood group incompatibility, however, only 5 percent are clinically affected, and the resulting anemia is usually mild. The condition differs from Rh CDE incompatibility in several respects. First, ABO incompatibility is often seen in firstborn infants, whereas sensitization to other blood group antigens is not. This is because most group O women have developed anti-A and anti-B isoagglutinins before pregnancy from exposure to bacteria displaying similar antigens. Second, ABO alloimmunization can affect future pregnancies, but unlike CDE disease, it rarely becomes progressively more severe. Last, most anti-A and anti-B antibodies are immunoglobulin M (IgM), which do not cross the placenta. Fetal red cells also have fewer A and B antigenic sites than adult cells and are thus less immunogenic. For these reasons, ABO alloimmunization is generally a pediatric disease rather than an obstetrical concern. There is no need to monitor for fetal hemolysis or to deliver the fetus early. Careful neonatal observation is essential, because hyperbilirubinemia may require treatment with phototherapy or occasionally transfusion (Chap. 33, p. 644).
Management of the Alloimmunized Pregnancy
An estimated 25 to 30 percent of fetuses from Rh D-alloimmunized pregnancies will have mild to moderate hemolytic anemia, and without treatment, up to 25 percent will develop hydrops (Tannirandorn, 1990). If alloimmunization is detected and the titer is below the critical value, the titer is generally repeated every 4 weeks for the duration of the pregnancy (American College of Obstetricians and Gynecologists, 2012). Importantly, however, if a woman has had a prior pregnancy complicated by alloimmunization, serial titer assessment is inadequate for surveillance of fetal anemia. In these instances, pregnancy is assumed to be at risk and is followed as discussed on page 310. Once a titer has reached a critical value, there is no benefit to repeating it. The pregnancy is at risk even if the titer decreases, and further evaluation is still required.
Determining Fetal Risk
The presence of maternal anti-D antibodies reflects her sensitization, but it does not necessarily indicate that the fetus will be affected or is even Rh D-positive. For example, in a non-Hispanic white couple in which the woman is Rh D-negative, there is an 85-percent chance that the man is Rh D-positive, but in 60 percent of these cases he will be heterozygous at the D-locus (American College of Obstetricians and Gynecologists, 2012). If he is heterozygous, then only half of his children will be at risk for hemolytic disease. Another consideration is that if a woman became sensitized in a prior pregnancy, her antibody titer might rise to high levels during the current pregnancy even if the current fetus is Rh D-negative. This is termed an amnestic response. Additionally, alloimmunization to a red cell antigen other than Rh D may have occurred following a blood transfusion in the past, and if that antigen is not present on paternal erythrocytes, the pregnancy is not at risk.
Initial evaluation of alloimmunization begins with determining the paternal erythrocyte antigen status. Provided that paternity is certain, if the father is negative for the red cell antigen to which the mother is sensitized, the pregnancy is not at risk. In an Rh D alloimmunized pregnancy in which the father is Rh D-positive, it is helpful to determine prenatal paternal zygosity for the Rh D antigen, and DNA-based analysis will establish this. If the father is heterozygous or if paternity is in question, the patient should be offered assessment of fetal antigen type. In the Unites States, this has traditionally been done with amniocentesis and PCR testing of uncultured amniocytes to assess fetal blood type (Chap. 13, p. 277) (American College of Obstetricians and Gynecologists, 2012). This test has a positive-predictive value of 100 percent and negative-predictive value of approximately 97 percent (Van den Veyver, 1996). Chorionic villus sampling is not generally performed because it is associated with greater risk for fetomaternal hemorrhage and may worsen alloimmunization. Fetal testing for other antigens is also available from reference laboratories using amniocentesis specimens. Examples include testing for E/e, C/c, Duffy, Kell, Kidd, and M/N.
More recently, noninvasive fetal Rh D blood typing has been performed using cell-free fetal DNA from maternal plasma (Chap. 13, p. 279). Accuracy is reported to be as high as 99 to 100 percent (Minon, 2008; Tynan, 2011). In a metaanalysis, only 3 percent of samples had inconclusive results (Geifman-Holtzman, 2006). Fetal Rh D blood typing with cell-free fetal DNA is routinely used in parts of Europe. There are two potential indications in Rh D negative pregnant women: (1) in women with Rh D alloimmunization, testing can identify fetuses who are also Rh D negative and do not require anemia surveillance, and (2) in women without Rh D alloimmunization, anti-D immune globulin might be withheld if the fetus is Rh D negative. However, concerns have been raised that use of the test to limit anti-D immune globulin administration could lead to an increased prevalence of Rh D alloimmunization (Goodspeed, 2013; Szczepura, 2011). As of 2013, cell-free fetal DNA testing for fetal Rh type has not been widely adopted in the United States.
Management of the alloimmunized pregnancy is individualized and may consist of maternal antibody titer surveillance, sonographic monitoring of the fetal MCA peak systolic velocity, amnionic fluid bilirubin studies, or fetal blood sampling. Accurate pregnancy dating is critical. The gestational age at which fetal anemia developed in prior pregnancies is important because anemia tends to occur earlier and be sequentially more severe.
Middle Cerebral Artery Doppler Velocimetry. In most specialized centers, serial measurement of the peak systolic velocity of the fetal middle cerebral artery has replaced amniocentesis for the detection of fetal anemia. The anemic fetus shunts blood preferentially to the brain to maintain adequate oxygenation. The velocity increases because of increased cardiac output and decreased blood viscosity (Moise, 2008a). The technique, which is discussed in Chapter 10 (p. 221), should be used only with adequate training and experience (American College of Obstetricians and Gynecologists, 2012).
In a landmark study, Mari and coworkers (2000) measured the MCA peak systolic velocity serially in 111 fetuses at risk for anemia and in 265 normal control fetuses. Threshold values > 1.5 multiples of the median (MoM) for given gestational ages correctly identified all fetuses with moderate or severe anemia. This provided a sensitivity of 100 percent, with a false-positive rate of 12 percent.
The MCA peak systolic velocity is followed serially, and values are plotted on a curve like the one shown in Figure 15-1. If the velocity is between 1.0 and 1.5 MoM and the slope is increasing—such that the value is approaching 1.5 MoM—surveillance is generally increased to weekly Doppler interrogation. If the MCA peak systolic velocity exceeds 1.5 MoM, further evaluation by fetal blood sampling is necessary to assess need for fetal transfusion. The false-positive rate increases significantly beyond 35 weeks, due to the normal increase in cardiac output that develops at this gestational age (Moise, 2008a; Zimmerman, 2002).
FIGURE 15-1 Doppler measurements of the peak systolic velocity in the middle cerebral artery in 165 fetuses at risk for severe anemia. The blue line indicates the median peak systolic velocity in normal pregnancies, and the red line shows 1.5 multiples of the median. (Redrawn from Oepkes, 2006, with permission.)
Amnionic Fluid Spectral Analysis. More than 50 years ago, Liley (1961) demonstrated the utility of amnionic fluid spectral analysis to measure bilirubin concentration. This permitted an estimate of hemolysis severity and indirect assessment of anemia. Amnionic fluid bilirubin is measured by a spectrophotometer and is demonstrable as a change in optical density absorbance at 450 nm—ΔOD450. The likelihood of fetal anemia is determined by plotting the ΔOD450 value on a graph that is divided into several zones. The original Liley graph is valid from 27 to 42 weeks’ gestation and contains three zones. Zone 1 indicates an Rh D-negative fetus or one with only mild disease. Zone 2 indicates fetal anemia, with hemoglobin concentrations of 11.0 to 13.9 g/dL for values in lower zone 2 and those of 8.0 to 10.9 g/dL for upper zone 2. Zone 3 indicates severe anemia, with hemoglobin concentration < 8.0 g/dL.
The Liley graph was subsequently modified by Queenan and associates (1993) to include gestational ages as early as 14 weeks (Fig. 15-2). The naturally high amnionic fluid bilirubin level at midpregnancy results in a large indeterminate zone. Here, bilirubin concentrations do not accurately predict fetal hemoglobin concentration. For this reason, if evaluation indicated that severe fetal anemia or hydrops before 25 weeks was likely, then fetal blood sampling was often performed.
FIGURE 15-2 Proposed amnionic fluid ΔOD450 management zones in pregnancies from 14 to 40 weeks. (Redrawn from Queenan, 1993, with permission.)
Middle cerebral artery velocimetry is noninvasive and does not confer the risks for pregnancy loss or increased alloimmunization associated with amniocentesis. Importantly, it is also more accurate than ΔOD450 assessment, particularly early in pregnancy. Oepkes and coworkers (2006) compared MCA Doppler velocimetry and amnionic fluid bilirubin studies. They found that MCA Doppler had significantly greater sensitivity and accuracy. For these reasons, amnionic fluid spectral analysis is currently used only when Doppler velocimetry is not readily available. It also may be considered if the MCA peak systolic velocity exceeds 1.5 MoM after 35 weeks’ gestation. In the latter situation, if ΔOD450 assessment indicates only mild hemolysis, delivery at 37 to 38 weeks has been recommended (American College of Obstetricians and Gynecologists, 2012; Moise, 2008b).
Fetal Blood Transfusion
If there is evidence of severe fetal anemia, either because of elevated MCA peak systolic velocity or development of fetal hydrops, management is strongly influenced by gestational age. The preterm fetus is usually evaluated with fetal blood sampling, as described in Chapter 14 (p. 300). Some recommend fetal transfusion until 30 to 32 weeks’ gestation and delivery at 32 to 34 weeks. To decrease neonatal morbidity from prematurity, others suggest intrauterine transfusion up to 36 weeks followed by delivery at 37 to 38 weeks (American College of Obstetricians and Gynecologists, 2012).
Intravascular transfusion into the umbilical vein under sonographic guidance is the preferred method of fetal transfusion. Peritoneal transfusion may be necessary with severe, early-onset hemolytic disease in the early second trimester, a time when the umbilical vein is too narrow to readily permit needle entry (Fox, 2008; Howe, 2007). With hydrops, although peritoneal absorption is impaired, some prefer to transfuse into both the peritoneal cavity and the umbilical vein.
Transfusion is generally recommended if the fetal hematocrit is < 30 percent. Once hydrops has developed, however, the hematocrit is generally 15 percent or lower. The red cells transfused are type O, Rh D negative, cytomegalovirus-negative, packed to a hematocrit of about 80 percent to prevent volume overload, irradiated to prevent fetal graft-versus-host reaction, and leukocyte-poor. The fetal-placental volume allows rapid infusion of a relatively large quantity of blood. Before transfusion, a paralytic agent such as vecuronium may be given to the fetus to minimize movements and potential needle-stick trauma. In a nonhydropic fetus, the target hematocrit is generally 40 to 50 percent. The volume transfused may be estimated by multiplying the estimated fetal weight in grams by 0.02 for each 10-percent increase in hematocrit needed (Giannina, 1998). In the severely anemic fetus, less blood is transfused initially, and another transfusion is planned for about 2 days later.
Subsequent transfusions usually take place every 2 to 4 weeks, depending on the hematocrit. The sensitivity of the MCA peak systolic velocity to detect severe anemia appears to be lower following an initial transfusion, such that it may not be reliable (Scheier, 2006). One schedule is to perform a second transfusion in 10 days, the third 2 weeks later, and any additional transfusions 3 weeks later (Moise, 2012). Following transfusion, the fetal hematocrit generally decreases by approximately 1 volume percent per day. A more rapid initial decline may be seen with hydropic fetuses.
Outcomes. Procedure-related complications have been reported in up to 9 percent of transfused pregnancies (van Kamp, 2005). These include fetal death in approximately 3 percent, neonatal death in about 2 percent, need for emergent cesarean delivery in 6 percent, and infection in 1 percent. Considering that fetal transfusion is potentially lifesaving in severely compromised fetuses, these risks should not dissuade therapy.
The overall survival rate following fetal transfusion approximates 90 percent (Lindenberg, 2013; Van Kamp, 2005). If transfusion is required before 20 weeks, survival rates are lower but may reach 80 percent at experienced centers (Canlorde, 2011; Lindenberg, 2013). Van Kamp and colleagues (2001) reported that if hydrops had developed, the survival rate approached 75 to 80 percent. However, of the nearly two thirds with resolution of hydrops following transfusion, more than 95 percent survived. The survival rate was below 40 percent if hydrops persisted.
Lindenberg and associates (2012) recently reviewed long-term outcomes following intrauterine transfusion in a cohort of more than 450 alloimmunized pregnancies. Alloimmunization was secondary to Rh D in 80 percent, Kell in 12 percent, and Rh c in 5 percent. Approximately a fourth of affected fetuses had hydrops, and more than half also required exchange transfusion in the neonatal period. The overall survival rate approximated 90 percent. Among nearly 300 children aged 2 to 17 years who participated in neurodevelopmental testing, fewer than 5 percent had severe impairments. These included severe developmental delay in 3 percent, cerebral palsy in 2 percent, and deafness in 1 percent.
Prevention of Rh D Alloimmunization
Anti-D immune globulin has been used for more than four decades to prevent Rh D alloimmunization and is one of the success stories of modern obstetrics. In countries without access to anti-D immune globulin, nearly 10 percent of Rh D-negative pregnancies are complicated by hemolytic disease of the fetus and newborn (Zipursky, 2011). With immunoprophylaxis, however, the alloimmunization risk is reduced to < 0.2 percent. Despite longstanding and widespread use, its mechanism of action is not completely understood.
As many as 90 percent of alloimmunization cases occur from fetomaternal hemorrhage at delivery. Routine postpartum administration of anti-D immune globulin to at-risk pregnancies within 72 hours of delivery decreases the alloimmunization rate by 90 percent (Bowman, 1985). Additionally, provision of anti-D immune globulin at 28 weeks’ gestation reduces the third-trimester alloimmunization rate from approximately 2 percent to 0.1 percent (Bowman, 1988).
Whenever there is doubt whether to give anti–D immunoglobulin, it should be given. Even if not needed, it will cause no harm, but failing to give it when needed can have severe consequences.
Current preparations of anti-D immune globulin are derived from human plasma donated by individuals with high-titer anti-D antibodies. Formulations prepared by cold ethanol fractionation and ultrafiltration can only be administered intramuscularly, because they contain plasma proteins that could result in anaphylaxis if given intravenously. However, newer formulations, prepared using ion exchange chromatography, may be administered either intramuscularly or intravenously. This is important for treatment of significant fetomaternal hemorrhage, which is discussed subsequently. Both preparation methods effectively remove viral particles, including hepatitis and human immunodeficiency viruses. Depending on the preparation, the half-life of anti-D immune globulin ranges from 16 to 24 days, which is why it is given both in the third trimester and following delivery. The standard intramuscular dose of anti-D immune globulin—300 μg or 1500 international units (IU)—will protect the average-sized mother from a fetal hemorrhage of up to 30 mL of fetal whole blood or 15 mL of fetal red cells.
In the United States, anti-D immune globulin is given prophylactically to all Rh D-negative, unsensitized women at approximately 28 weeks, and a second dose is given after delivery if the infant is Rh D-positive (American College of Obstetricians and Gynecologists, 2010). Before the 28-week dose of anti-D immune globulin, repeat antibody screening is recommended to identify individuals who have become alloimmunized (American Academy of Pediatrics and American College of Obstetricians and Gynecologists 2012). Following delivery, anti-D immune globulin should be given within 72 hours. Importantly, if immune globulin is inadvertently not administered following delivery, it should be given as soon as the omission is recognized, because there may be some protection up to 28 days postpartum (Bowman, 2006). Anti-D immune globulin is also administered after pregnancy-related events that could result in fetomaternal hemorrhage (see Table 15-2).
Anti-D immune globulin may produce a weakly positive—1:1 to 1:4—indirect Coombs titer in the mother. This is harmless and should not be confused with development of alloimmunization. Additionally, as the body mass index increases above 27 to 40 kg/m2, serum antibody levels decrease by 30 to 60 percent and may be less protective (MacKenzie, 2006; Woelfer, 2004). Rh D-negative women who receive other types of blood products—including platelet transfusions and plasmapheresis—are also at risk of becoming sensitized, and this can be prevented with anti-D immune globulin. Rarely, a small amount of antibody crosses the placenta and results in a weakly positive direct Coombs test in cord and infant blood. Despite this, passive immunization does not cause significant fetal or neonatal hemolysis.
In approximately 1 percent of pregnancies, the volume of fetomaternal hemorrhage exceeds 30 mL of whole blood (Ness, 1987). A single dose of anti-D immune globulin would be insufficient in such situations. If additional anti-D immune globulin is considered only for women with risk factors—examples include abdominal trauma, placental abruption, placenta previa, intrauterine manipulation, multifetal gestation, or manual placenta removal—half of those who require more than the 1500-IU dose may be missed. Because of these observations, the American Association of Blood Banks recommends that all D-negative women be tested at delivery with a rosette test or Kleihauer-Betke test (Snyder, 1998).
The rosette test is used to identify whether fetal Rh D-positive cells are present in the circulation of an Rh D-negative woman. It is a qualitative test. A sample of maternal blood is mixed with anti-D antibodies that coat any Rh D-positive fetal cells present in the sample. Indicator red cells bearing the D-antigen are then added, and rosettes form around the fetal cells as the indicator cells attach to them by the antibodies. Thus, if rosettes are visualized, there are fetal Rh D-positive cells in that sample.
The Kleihauer-Betke test is a quantitative test used either in the setting of Rh D incompatibility or any time a large fetomaternal hemorrhage is suspected—regardless of antigen status. It is discussed on page 313.
The dosage of anti-D immune globulin is calculated from the estimated volume of the fetal-to-maternal hemorrhage, as described on page 311. One 1500-IU (300 μg) ampule is given for each 15 mL of fetal red cells or 30 mL of fetal whole blood to be neutralized. If using an intramuscular preparation of anti-D immune globulin, no more than five doses may be given in a 24-hour period. If using an intravenous preparation, two ampules—totaling 3000 IU (600 μg)—may be given every 8 hours. To determine if the administered dose was adequate, the indirect Coombs test may be performed. A positive result indicates that there is excess anti–D immunoglobulin in maternal serum, thus demonstrating that the dose was sufficient. Alternatively, a rosette test may be performed to assess whether circulating fetal cells remain.
Weak D Antigens
Women who are positive for weak D antigens, formerly called Du, are not considered at risk for hemolytic disease and do not require anti-D immune globulin (American College of Obstetricians and Gynecologists, 2010). There are, however, D-antigen variants—termed partial D antigens—that can result in Rh D alloimmunization and cause hemolytic disease (Daniels, 2013). If a D-negative woman delivers a weak D-positive infant, she should be given anti-D immune globulin. It is worth emphasizing that if there is any doubt regarding D-antigen status, then immune globulin should be given.
It is likely that all pregnant women experience a small fetomaternal hemorrhage, and in two-thirds, this may be sufficient to provoke an antigen-antibody reaction. As shown in Figure 15-3, the incidence increases with gestational age, as does the volume of fetal blood in the maternal circulation. Large volumes of blood loss—true fetomaternal hemorrhage—are fortunately rare. In a series of more than 30,000 pregnancies, de Almeida and Bowman (1994) found evidence of fetomaternal hemorrhage > 80 mL in approximately 1 per 1000 births, and hemorrhage > 150 mL in 1 per 5000 births.
FIGURE 15-3 Incidence of fetal-to-maternal hemorrhage during pregnancy. The numbers at each data point represent total volume of fetal blood estimated to have been transferred into the maternal circulation. (Data from Choavaratana, 1997.)
Fetomaternal hemorrhage may follow maternal trauma, may occur with placenta previa or vasa previa, and may follow amniocentesis or external cephalic version (Giacoia, 1997; Rubod, 2007). In more than 80 percent of cases, however, no cause is identified. With significant hemorrhage, the most common presenting complaint is decreased fetal movement (Eichbaum, 2006; Hartung, 2000; Wylie, 2010). A sinusoidal fetal heart rate pattern, although uncommon, is occasionally seen and warrants immediate evaluation (Chap. 24, p. 482). Sonography may demonstrate elevated MCA peak systolic velocity, and hydrops may be identified (Eichbaum, 2006; Giacoia, 1997; Hartung, 2000). If fetomaternal hemorrhage is suspected based on either a sinusoidal fetal heart rate pattern or positive Kleihauer-Betke test result, then the finding of an elevated MCA peak systolic velocity or hydrops should prompt consideration of either fetal transfusion or delivery.
One limitation of quantitative tests for fetal cells in the maternal circulation is that they do not provide information regarding hemorrhage timing or chronicity (Wylie, 2010). In general, anemia developing gradually or chronically, as in alloimmunization, is better tolerated by the fetus than acute anemia. Chronic anemia may not produce fetal heart rate abnormalities until the fetus is moribund. In contrast, significant acute hemorrhage is poorly tolerated by the fetus and may cause profound fetal neurological impairment from cerebral hypoperfusion, ischemia, and infarction. In some cases, fetomaternal hemorrhage is identified during stillbirth evaluation (Chap. 35, p. 664).
Tests for Fetomaternal Hemorrhage
Once fetomaternal hemorrhage is recognized, the volume of fetal blood loss can be estimated. The volume may influence obstetrical management and is essential to determining the appropriate dose of anti D-immune globulin if the woman is Rh D-negative.
The most commonly used quantitative test for fetal red cells in the maternal circulation is the acid elution or Kleihauer-Betke (KB) test (Kleihauer, 1957). Fetal erythrocytes contain hemoglobin F, which is more resistant to acid elution than hemoglobin A. After exposure to acid, only fetal hemoglobin remains, such that after staining, the fetal erythrocytes appear red and adult cells appear as “ghosts” (Fig. 15-4). Fetal cells are counted and expressed as a percentage of adult cells. The test is labor intensive. Moreover, it may be less accurate in two situations: (1) cases of maternal hemoglobinopathy in which maternal red cells carry excess fetal hemoglobin and (2) cases near or at term, at which time the fetus has already started to produce hemoglobin A.
FIGURE 15-4 Kleihauer-Betke test demonstrating massive fetal-to-maternal hemorrhage. After acid-elution treatment, fetal red cells rich in hemoglobin F stain darkly, whereas maternal red cells with only very small amounts of hemoglobin F stain lightly.
The hemorrhaged fetal blood volume is calculated from the Kleihauer-Betke test result using the following formula:
One method is to estimate the maternal blood volume (MBV) as 5000 mL for a normal-size, normotensive women at term. Thus, for 1.7-percent positive KB-stained cells in a woman of average size with a hematocrit of 35 percent giving birth to a term infant weighing 3000 g and whose hematocrit is 50 percent:
The fetal-placental blood volume at term approximates 125 mL/kg. For this 3000-g fetus, that would equate to 375 mL. Thus, this fetus has lost approximately 15 percent (60 ÷ 375 mL) of the fetal-placental volume. Because the hematocrit is 50 percent in a term fetus, this 60 mL of whole blood represents 30 mL of red cells lost over time into the maternal circulation. This loss should be well tolerated hemodynamically but would require two 300-μg doses of anti-D immunoglobulin to prevent alloimmunization. A more precise method to estimate the maternal blood volume includes a calculation based on the maternal height, weight, and anticipated blood volume increase due to gestational age (Chap. 41, p. 781).
Fetomaternal hemorrhage can also be quantified using flow cytometry to measure red cell size (Dziegiel, 2006). This is an automated test and is unaffected by maternal levels of fetal hemoglobin F or by fetal levels of hemoglobin A. In direct comparison studies with the Kleihauer-Betke test, flow cytometry has been reported to be more sensitive and accurate (Chambers, 2012; Fernandes, 2007).
This condition is also referred to as neonatal alloimmune thrombocytopenia (NAIT) or fetal and neonatal alloimmune thrombocytopenia (FNAIT). Alloimmune thrombocytopenia (AIT) is the most common cause of severe thrombocytopenia among term newborns, with a frequency of 1 to 2 per 1000 (Kamphuis, 2010; Pacheco, 2013; Risson, 2012). AIT is caused by maternal alloimmunization to paternally inherited fetal platelet antigens. The resulting maternal antiplatelet antibodies cross the placenta in a manner similar to red cell alloimmunization (p. 306). Unlike immune thrombocytopenia, the maternal platelet count is normal. And unlike Rh-D alloimmunization, severe sequelae may affect the first at-risk pregnancy.
Maternal platelet alloimmunization is most commonly against human platelet antigen-1a (HPA-1a). This is followed by HPA-5b, HPA-1b, and HPA-3a, and alloimmunization to other antigens accounts for only 1 percent of reported cases. Alloimmunization to HPA-1a accounts for 80 to 90 percent of cases and is associated with the greatest severity (Bussel, 1997; Knight, 2011; Tiller, 2013).
Approximately 85 percent of white individuals are positive for HPA-1a. Two percent are homozygous for HPA-1b and thus are at risk for alloimmunization. But, only 10 percent of homozygous HPA-1b mothers carrying an HPA-1a fetus will produce anti-platelet antibodies. Approximately a third of affected fetuses or neonates will develop severe thrombocytopenia, and 10 to 20 percent with severe thrombocytopenia sustain an intracranial hemorrhage (ICH) (Kamphuis, 2010). As a result, population-based screening studies have identified FNAIT-associated ICH in 1 per 25,000 to 60,000 pregnancies (Kamphuis, 2010; Knight, 2011).
FNAIT has a broad spectrum of presentation. In some, neonatal thrombocytopenia may be an incidental finding or the infant may present with petechiae. Alternatively, the fetus or neonate may develop devastating ICH—often before birth. Of 600 pregnancies with alloimmune thrombocytopenia identified through a large international registry, fetal or neonatal ICH complicated 7 percent of cases (Tiller, 2013). Hemorrhage affected the first-born child in 60 percent and occurred before 28 weeks’ gestation in half. A third of affected children died soon after birth, and 50 percent of survivors had severe neurological disabilities. Bussel and coworkers (1997) evaluated fetal platelet counts before therapy in 107 fetuses with FNAIT. Thrombocytopenia severity was predicted by a prior sibling with perinatal ICH, and 98 percent of cases were identified this way. The initial platelet count was < 20,000/mL in 50 percent. In cases in which the platelet count was initially > 80,000/mL, they noted that it dropped by more than 10,000/mL each week without therapy.
Diagnosis and Management
The diagnosis of alloimmune thrombocytopenia is usually made after the first affected pregnancy in a woman with a normal platelet count whose neonate is found to have unexplained severe thrombocytopenia. Rarely, the diagnosis is ascertained after finding fetal ICH. The condition recurs in 70 to 90 percent of subsequent pregnancies, is often severe, and usually develops earlier with each successive pregnancy. Traditionally, fetal blood sampling was performed to detect fetal thrombocytopenia and tailor therapy, and platelets were transfused if the fetal platelet count was < 50,000/mL. But, concern with procedure-related complications has led experts to recommend abandoning routine fetal platelet sampling in favor of empiric treatment with intravenous immune globulin (IVIG) and prednisone (Berkowitz, 2006; Pacheco, 2011).
Therapy is stratified according to whether a prior affected pregnancy was complicated by perinatal ICH, and if so, at what gestational age (Table 15-3). Pioneering work by Bussel (1996) and Berkowitz (2006) and their colleagues demonstrated the efficacy of such treatment. In one series of 50 pregnancies with fetal thrombocytopenia secondary to FNAIT, IVIG resulted in an increased platelet count of approximately 50,000/mL, and no fetus developed ICH (Bussel, 1996). Among pregnancies at particularly high risk—based on platelet count < 20,000/mL or sibling with FNAIT-associated ICH—the addition of corticosteroids to IVIG increased the platelet count in 80 percent of cases (Berkowitz, 2006). Cesarean delivery has been recommended at or near term. A noninstrumental vaginal delivery may be considered only if fetal blood sampling has demonstrated a platelet count > 100,000/mL (Pacheco, 2011).
TABLE 15-3. Fetal-Neonatal Alloimmune Thrombocytopenia (FNAIT) Treatment Recommendations
Additional considerations include risks and costs associated with therapy. Side effects of IVIG may include fever, headache, nausea/vomiting, myalgia, and rash. Maternal hemolysis also has been described (Rink, 2013). As of 2011, costs for various preparations of IVIG were approximately $70 per gram or nearly $10,000 for each weekly 2-g/kg infusion for an average-size pregnant woman (Pacheco, 2011).
In pregnant women with immune thrombocytopenia (ITP), autoimmune antiplatelet IgG antibodies may cross the placenta and cause fetal thrombocytopenia. Maternal ITP is discussed in Chapter 56 (p. 1114). Fetal thrombocytopenia is usually mild. However, neonatal platelet levels may fall rapidly after birth and reach a nadir at 48 to 72 hours of life. Neither the maternal platelet count or identification of antiplatelet antibodies, nor treatment with corticosteroids is predictive of fetal or neonatal platelet counts. Importantly, fetal platelet counts are usually adequate to allow vaginal delivery without an increased risk of ICH. Fetal bleeding complications are considered rare, and fetal blood sampling is not recommended (Neunert, 2011). Delivery mode is based on standard obstetrical indications.
The term hydrops refers to excessive accumulation of serous fluid in the body, and strictly defined, hydrops fetalis is edema of the fetus. Traditionally, the diagnosis was made after delivery of a massively edematous neonate, often stillborn (Fig. 15-5). With sonography, hydrops has become a prenatal diagnosis. It is defined as two or more fetal effusions—pleural, pericardial, or ascites—or one effusion plus anasarca. With condition progression, edema is invariably a component, often accompanied by placentomegaly and hydramnios. Hydrops may result from a wide range of conditions with varying pathophysiologies, each with the potential to make the fetus severely ill. It is divided into two categories. If found in association with red cell alloimmunization, it is termed immune, otherwise, it is nonimmune.
FIGURE 15-5 Hydropic, macerated stillborn infant and characteristically large placenta. The etiology was B19 parvovirus infection. (Photograph contributed by Dr. April Bleich.)
The incidence of immune hydrops has dramatically decreased with the advent of anti-D immune globulin, MCA Doppler studies for detection of severe anemia, and prompt fetal transfusion when needed. Only an estimated 10 percent of hydrops cases are caused by red cell alloimmunization (Bellini, 2009, 2012; Santolaya, 1992).
The pathophysiology underlying hydrops remains unknown. Immune hydrops is postulated to share several physiological abnormalities with nonimmune hydrops. As shown in Figure 15-6, these include decreased colloid oncotic pressure, increased hydrostatic (or central venous) pressure, and increased vascular permeability. Immune hydrops results from transplacental passage of maternal antibodies that destroy fetal red cells. Resultant anemia stimulates marrow erythroid hyperplasia and extramedullary hematopoiesis in the spleen and liver. The latter likely causes portal hypertension and impaired hepatic protein synthesis, which decreases plasma oncotic pressure (Nicolaides, 1985). Fetal anemia also may raise central venous pressure (Weiner, 1989). Finally, tissue hypoxia from anemia may increase capillary permeability, such that fluid collects in the fetal thorax, abdominal cavity, and/or subcutaneous tissue.
FIGURE 15-6 Proposed pathogenesis of immune and nonimmune hydrops fetalis. (Adapted from Bellini, 2009; Lockwood, 2009.)
The degree of anemia in immune hydrops is typically severe. Nicolaides and associates (1988) reported that the hemoglobin concentration was 7 to 10 g/dL below the normal mean for gestational age in a cohort of 48 fetuses with immune hydrops. Similarly, in a series of 70 pregnancies with fetal anemia from red cell alloimmunization, Mari and coworkers (2000) found that all those with immune hydrops had hemoglobin values below 5 g/dL. As discussed on page 310, immune hydrops is treated with fetal blood transfusions (van Kamp, 2001).
Currently, nearly 90 percent of cases of hydrops are nonimmune (Bellini, 2009, 2012; Santolaya, 1992). The prevalence estimate is 1 per 1500 second-trimester pregnancies (Heinonen, 2000). The number of specific disorders that can lead to nonimmune hydrops is extensive. Etiologies and the proportion of births within each hydrops category from a review of more than 5400 affected pregnancies are summarized in Table 15-4. A cause is identified in at least 60 percent prenatally and in more than 80 percent postnatally (Bellini, 2009; Santo, 2011). As shown in Figure 15-6, there are several different pathophysiological processes proposed to account for the final common pathway of hydrops fetalis.
TABLE 15-4. Some Etiologies of Nonimmune Hydrops Fetalis
Importantly, the etiology of nonimmune hydrops varies according to when in gestation it is identified. Of those diagnosed prenatally, aneuploidy accounts for approximately 20 percent, cardiovascular abnormalities for 15 percent, and infections for 14 percent—the most common of these being parvovirus B19 (Santo, 2011). Overall, only 40 percent of pregnancies with nonimmune hydrops result in a liveborn neonate. For these, the neonatal survival rate is only about 50 percent. Sohan and colleagues (2001) reviewed 87 pregnancies with hydrops and found that 45 percent of those diagnosed before 24 weeks’ gestation had a chromosomal abnormality. The most common was 45, X—Turner syndrome (Chap. 10, p. 205), and in such cases, the survival rate was < 5 percent. If hydrops is detected in the first trimester, the aneuploidy risk is nearly 50 percent, and most have cystic hygromas (Fig. 10-16, p. 206) (Has, 2001).
Thus, the prognosis of nonimmune hydrops is guarded but is heavily dependent on etiology. In large series from Thailand and Southern China, α4-thalassemia is the predominant cause of nonimmune hydrops, accounting for 30 to 50 percent of cases and conferring an extremely poor prognosis (Liao, 2007; Ratanasiri, 2009; Suwanrath-Kengpol, 2005). In contrast, Sohan and associates (2001) found that treatable causes of nonimmune hydrops—parvovirus, chylothorax, and tachyarrhythmias—each comprised about 10 percent of cases, and with fetal therapy, two thirds of fetuses with these etiologies survived.
Hydrops is readily detected sonographically. As noted, two effusions or one effusion plus anasarca are required for diagnosis. Edema may be particularly prominent around the scalp, or equally obvious around the trunk and extremities. Effusions are visible as fluid outlining the lungs, heart, or abdominal viscera (Fig. 15-7).
FIGURE 15-7 Hydropic features. A. This profile of a 23-week fetus with nonimmune hydrops secondary to B19 parvovirus infection depicts scalp edema (arrowheads) and ascites (*). B. In this coronal image, prominent pleural effusions (*) outline the lungs (L). This 34-week fetus had hydrops secondary to an arteriovenous malformation in the brain, known as a vein of Galen aneurysm. Fetal ascites is also present (arrows), as is anasarca. C. This axial (transverse) image depicts a pericardial effusion (arrows) in a 23-week fetus with hydrops from B19 parvovirus infection. The degree of cardiomegaly is impressive, and the ventricular hypertrophy raises concern for myocarditis, which can accompany parvovirus infection. D. This axial (transverse) image depicts fetal ascites (*) in a 15-week fetus with hydrops secondary to large cystic hygromas. Anasarca is also seen (brackets).
In many cases, targeted sonographic and laboratory evaluation will identify the underlying cause of fetal hydrops. These include cases due to fetal anemia, arrhythmia, structural abnormality, aneuploidy, placental abnormality, or complications of monochorionic twinning. Depending on the circumstances, initial evaluation includes the following:
1. Indirect Coombs test for alloimmunization
2. Targeted sonographic fetal and placental examination, including:
• A detailed anatomic survey to assess for the structural abnormalities listed in Table 15-4
• MCA Doppler velocimetry to assess for fetal anemia
• Fetal echocardiography with M-mode evaluation
3. Amniocentesis for fetal karyotype and for B19 parvovirus, cytomegalovirus, and toxoplasmosis testing as discussed in Chapter 64. Consideration of chromosomal microarray analysis if fetal anomalies are present
4. Consideration of Kleihauer-Betke test for fetomaternal hemorrhage if anemia is suspected, depending on findings and test results
5. Consideration of testing for α-thalassemia and/or inborn errors of metabolism.
Isolated Effusion or Edema. Although one effusion or anasarca alone is not diagnostic for hydrops, the above evaluation should be considered if these are encountered, as hydrops may develop. For example, an isolated pericardial effusion may be the initial finding in fetal B19 parvovirus infection (Chap. 64, p. 1244). An isolated pleural effusion may represent a chylothorax, which is amenable to prenatal diagnosis, and for which fetal therapy may be lifesaving if hydrops develops (Chap. 16, p. 329). Isolated ascites also may be the initial finding in fetal B19 parvovirus infection or it may result from a gastrointestinal abnormality such as meconium peritonitis. Finally, isolated edema, particularly involving the upper torso or the dorsum of the hands and feet, may be found in Turner or Noonan syndrome or may represent congenital lymphedema syndrome (Chap. 13, p. 264).
An association between fetal hydrops and development of maternal edema in which the fetus mirrors the mother is attributed to Ballantyne. He called the condition triple edema because the fetus, mother, and placenta all became edematous. The etiology of the hydrops is not related to development of mirror syndrome. It has been associated with hydrops from Rh D alloimmunization, twin-twin transfusion syndrome, placental chorioangioma, and with fetal cystic hygroma, Ebstein anomaly, sacrococcygeal teratoma, chylothorax, bladder outlet obstruction, supraventricular tachycardia, vein of Galen aneurysm, and various congenital infections (Braun, 2010).
In a review of more than 50 cases of mirror syndrome, Braun and coworkers (2010) found that approximately 90 percent of women had edema, 60 percent had hypertension, 40 percent had proteinuria, 20 percent had liver enzyme elevation, and nearly 15 percent had headache and visual disturbances. Based on these findings, it is reasonable to consider mirror syndrome a form of severe preeclampsia (Espinoza, 2006; Midgley, 2000). Others, however, have suggested that it is a separate disease process with hemodilution rather than hemoconcentration (Carbillon, 1997; Livingston, 2007). There have been recent reports describing the same imbalance of angiogenic and antiangiogenic factors observed with preeclampsia and thus supporting a common pathophysiology (Espinoza, 2006; Goa, 2013; Llurba, 2012). These findings, which include elevated concentrations of soluble fms-like tyrosine kinase-1 (sFlt-1), decreased placental growth factor (PlGF) levels, and elevation of soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) concentrations, are discussed further Chapter 40 (p. 735).
In most cases with mirror syndrome, prompt delivery is indicated and followed by resolution of maternal edema and other findings in approximately 9 days (Braun, 2010). There are, however, isolated cases of fetal anemia, supraventricular tachycardia, hydrothorax, and bladder outlet obstruction in which successful fetal treatment resulted in resolution of both fetal hydrops and maternal mirror syndrome (Goa, 2013; Livingston, 2007; Llurba, 2012; Midgley, 2000). In two of these cases, normalization of the angiogenic imbalance also occurred following fetal transfusion for B19 parvovirus infection (Goa, 2013; Llurba, 2012). Fetal therapy for these conditions is reviewed in Chapter 16. Given the parallels to severe preeclampsia, delaying delivery to effect fetal therapy should be considered only with caution. If the maternal condition deteriorates, delivery is recommended.
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