Mechanism

The pathophysiology of immune hydrops is exemplified by RhD isoimmunization. However, several other RBC antigens have been linked to the development of immune hydrops ( Table 17-1 ). The RhD antigen is one of the most immunogenic antigens expressed on the surface of RBCs ; other antigens differ in their ability to cause the disease. Maternal alloimmunization results from the exposure to foreign RhD-positive cells that eventually lead to an immunologic response. The most common cause of this phenomenon is a transplacental passage of fetal cells into maternal circulation, such as during a miscarriage, amniocentesis, trauma, or delivery ( Fig. 17-9 ). Using Kleihauer-Betke stain, as many as 75% of women have some evidence of the presence of fetal RBCs in maternal circulation during pregnancy or delivery.

A, Initial pregnancy in which the mother is Rh negative and is carrying a fetus that is Rh positive. B, At the time of delivery, the fetal Rh-positive red blood cells enter the maternal circulation and initiate an antibody response. C, In a subsequent pregnancy, antibodies enter the fetal circulation, attaching to the red blood cells and resulting in the sequestration and destruction of these cells.

(Illustration by James A. Cooper, MD, San Diego, CA.)

The risk of fetal-maternal hemorrhage also increases with gestational age: 0.01 mL of fetal blood may be detected in 3% of pregnancies the first trimester and 46% during the third trimester. The probability of alloimmunization is directly proportional to the amount of fetal blood that crosses the placental barrier. It has been estimated that 0.25 mL of fetal blood is the minimum amount required to lead to alloimmunization. For these reasons, delivery poses the greatest risk for Rh sensitization. The proper administration of RhD immune globulin, however, can effectively prevent the formation of maternal antibodies. Transfusion of incompatible blood can also lead to maternal sensitization; this is the most common mechanism explaining alloimmunization from other RBC antigens.

The genetics of the Rh system are complex. The Rh factor is among the largest of the blood groups and contains well over 50 different recognized antigens. Rearrangements in the Rh locus can result in hybrid proteins that can lead to additional antigen expression. Only a small number of these antigens are actually clinically relevant. During pregnancy there are three antigens that can lead to the formation of antibodies and alloimmunization, Rh (D) and Rh (CcEe). These antigens are encoded by two genes located on the short arm of chromosome 1, the RhD and RhCE genes. Each gene contains 10 exons. It has been speculated that through a mechanism involving alternative splicing these two genes could direct the synthesis of different proteins corresponding to different variations in Rh antigens. Fischer and Race have proposed a concept to explain the inheritance of the alleles in the Rh genes. In the majority of patients, three pairs of Rh antigens are inherited from each parent—Cc, Dd, and Ee. An individual can be heterozygous or homozygous for each allele. The Rh-positive or Rh-negative status of a patient is determined by the presence or absence (or possible nonexpression) of the D antigen site. The Rh (CcEe) has fewer alleles and a limited number of mutations reported. The Cc and the Ee alleles are codominant and expressed in a heterozygous fashion. It should also be noted that the symbol “d” simply indicates the absence of D antigen; there is no “d” allele.

The D antigen is a transmembrane protein located on the surface of the erythrocyte that contains several extracellular loops. More than 150 different alleles for the Rh (D) gene have been reported, most likely a byproduct of numerous mutations within the gene. The complete absence of the RhD antigen is called a negative allele. The gene frequency varies according to race and ethnicity. Rh-negative individuals are reported in approximately 1% of the Chinese and Japanese populations, whereas the Basque tribe in northern Spain has a reported incidence of approximately 30%. In the patient population of the Americas, the difference among races and various ethnic backgrounds is also noticeable. Caucasians of European descent have an Rh-negative incidence of 15%, whereas Hispanics and African Americans have an incidence closer to 8%. Among several African populations, a variant of Rh (D) gene referred to as the RhD pseudogene (Rh ψ) has also been described. This gene contains all the 10 exons normally found on the Rh (D) gene but has a difference in gene expression due to the presence of a stop codon in the intron between exons 3 and 4. Because of this change, the Rh (D) protein is not synthesized, and the patient is phenotypically Rh (D) negative. This gene is detected in more than 60% of Rh-negative black Africans.

After the first exposure to the D antigen, a weak immunologic response is mounted over 6 weeks to a year. The predominant immunoglobulin produced in this first interaction is immunoglobulin (Ig) M. This large protein does not cross the placenta, and therefore the first pregnancy is usually not affected. However, a second exposure to the antigen can cause B lymphocytes to differentiate into plasma cells and proliferate. This amnestic response in the immune system is characterized by the production of IgG almost entirely; these IgG antibodies can cross the placental barrier. Two IgG subclasses have been shown to be produced during this stage, IgG1 and IgG3. Disease severity appears to be lower in cases in which IgG1 is almost exclusively present.

Fetal erythropoiesis begins in the yolk sac by day 21. The Rh antigen can be expressed as early as day 30 of gestation. After the second exposure, there is an increase in the production of IgG antibodies leading to an increase in maternal titers. These antibodies recognize and attach to the foreign antigen on the fetal RBC surface. The IgG antibody lacks the ability to bind complement, and therefore, the antibody-coated fetal RBCs are sequestered by macrophages and are destroyed by the reticuloendothelial system extravascularly. This chain of events leads to the development of fetal anemia.

As the life span of the fetal RBC is reduced, a greater burden is placed on the hematopoietic system. It has been proposed that medullary hematopoiesis is stimulated in the early stages of mild anemia and that recruitment of extramedullary sites occurs only after anemia has progressed and become severe. This is evidenced by the fact that in severe disease, both the liver and the spleen become enlarged because of congestion and increased extramedullary hematopoiesis, causing the release of nucleated RBC precursors (erythroblasts). In a study evaluating umbilical cord samples from 127 pregnancies, Nicolaides and associates showed that significant erythroblastosis was present only when the hemoglobin deficit was greater than 7 g/dL ( Fig. 17-10 ). Severe anemia can lead to decreased oxygen delivery and hypoxia in certain end organs, causing cardiac output to increase as a compensatory mechanism. However, these mechanisms may be inadequate to maintain adequate perfusion. The loss of the buffer function of fetal RBCs also contributes to worsening acid-base disturbances. Soothill and colleagues reported an increase in the lactate concentration in the umbilical artery when hemoglobin levels were below 8 g/dL and an increase in umbilical vein concentration when this level dropped to 4 g/dL. This finding implies that disease progression leads to a shift in cellular metabolism to a predominantly anaerobic state.

Relationship between fetal hemoglobin and gestational age.

(From Mari G, Deter RL, Carpenter RL, et al: Noninvasive diagnosis by Doppler ultrasonography of fetal anemia due to maternal red-cell alloimmunization. Collaborative Group for Doppler Assessment of the Blood Velocity in Anemic Fetuses (Level II-1). N Engl J Med 342:9-14, 2000; used with permission.)

The exact mechanism leading to fetal hydrops is incompletely understood. It is well known that anemia must be severe before hydrops develops and that fetal hemoglobin must fall below six standard deviations from the mean for that gestational age. Initial studies indicated that low fetal albumin levels leading to decreased colloid pressure and increased extravasation could be a contributing factor leading to hydrops. However, more recent studies have shown that hypoalbuminemia is found in only a small percentage of fetuses with hydrops and that drops in albumin levels appear to be a component of the very late stages of the disease. Other factors may also lead to increased vascular permeability facilitating the development of hydrops. Berger and coworkers demonstrated that concentration of ferritin and lipid-peroxidation products were increased, leading to the suggestion that iron overload and free radical damage may contribute to the pathogenesis. Cardiac dysfunction is also common among fetuses affected by severe anemia and could possibly play a role in the disease process. Affected fetuses develop an increase in both biventricular cardiac diameter and umbilical venous pressure. The increase in venous pressure may potentially lead to increased fluid extravasation, favoring the development of hydrops. The increase in venous pressure could also lead to an obstruction of lymphatic flow. Interestingly enough, it has also been shown that umbilical venous pressure decreases as early as 24 hours following therapeutic fetal intravascular transfusion.

Management

Severe fetal anemia that eventually leads to hydrops has direct implications for both fetal survival and long-term neonatal and infant outcomes. Cases of fetal anemia characterized by severe hydrops have a 74% survival rate compared to a 94% survival rate seen in nonhydrops cases. In a longitudinal study evaluating the long-term neurologic outcomes following intrauterine transfusion for RBC alloimmunization, over 95% of the 291 children followed were found to have normal developmental outcomes. However, a history of hydrops was found to be a significant risk factor for neurodevelopmental impairment.

Prevention is the key to management of alloimmunization. This starts at the first prenatal visit; a detailed history should be obtained from all patients. Specifics and details on past obstetric events as well as any previous transfusion of blood products are crucial to determining patients who may be at risk. Initial laboratory workup should include ABO typing with determination of RhD status as well as an antibody screen via a maternal indirect Coombs test. If a patient is found to be RhD negative, it should be determined if she is a candidate for prophylactic Rh immunoglobulin. If the antibody screen is positive, the blood bank should report the identity and the titer of the antibody. Titer levels are used to assess the likelihood of risk that the fetus may become affected. Antibodies that have been associated with hemolytic disease of the newborn are listed in Table 17-1 .

A repeat antibody screen for RhD should be done at 24 to 28 weeks. If this screen is negative, the patient is a candidate for anti-D prophylaxis via passive immunoglobulin administration. Although the exact mechanism of action is unknown, this strategy has been shown to be effective. In the United States, it is recommended that a single dose of 300 µg of anti-D immunoglobulin be administered at 28 weeks’ gestation. Immune prophylaxis probably works through the antigen clearance hypothesis. In this scenario, the administered immunoglobulin attaches to the RBCs. This interaction favors the removal of the coated cells by the mononuclear phagocytic system, particularly the macrophages, therefore not allowing these antigens to be recognized by the immune system. However, recent studies using several different animal models have shown that this hypothesis does not explain the entire mechanism of action. Other antigen-presenting cells, capable of initiating an immune response, also play a role in the clearance of D-positive RBCs.

After the administration of the immune prophylaxis at 28 weeks’ gestation, some experts recommend that a second dose of 300 µg be administered if the woman has not delivered within 12 weeks of her dose. One prophylactic dose of 300 µg of anti-D immunoglobulin is enough to prevent exposure produced by 30 mL of RhD-positive blood or 15 mL of fetal cells. Current guidelines dictate that if an RhD-negative woman delivers an RhD-positive infant, she should have a quantitative test (i.e., Kleihauer-Betke stain) done to determine the exact amount of anti-D immune globulin to be administered. The result of this test is typically multiplied by a factor of 50 to determine the total amount of fetomaternal hemorrhage. The dose should be administered within 72 hours of delivery, although this strategy has been shown to be effective up to 28 days after delivery. Also, women at risk for RhD alloimmunization should also receive a dose of prophylactic immunoglobulin after any obstetric event or procedure that puts her at risk for maternal-fetal hemorrhage.

Women who have been sensitized against RhD should have maternal antibody titer levels repeated every 4 weeks during the first and second trimesters and every 2 weeks during the third trimester in order to determine the risk of developing hemolytic disease. Maternal-fetal ABO incompatibility may be protective and decreases the risk of D alloimmunization. If a fetus is both RhD positive and ABO incompatible with the mother, this reduces the risk of alloimmunization to about 2%. Variations in the techniques used to determine the antibody level have led to the use of different thresholds to determine when a fetus is at risk for developing anemia. The titer value that is determined to be critical varies from 1 : 8 to 1 : 32. Clinicians should be familiar with the reference value used within their facilities.

Paternal testing can also be used to determine the risk of developing fetal anemia. RhD-positive fathers have a probability of being heterozygous. In the past, mathematical modeling along with ethnicity was used to determine the probability of heterozygosity. Advances in genetic testing have now allowed for the direct determination of paternal zygosity. If paternity is assured, this test can be used in the management algorithm of an RhD-negative woman. If the father is found to be heterozygous, the fetus has a 50% chance of being RhD negative. If the father is RhD negative, the fetus will likewise be RhD negative and therefore will not be at risk.

It has been estimated that 40% of RhD-negative women carry an RhD-negative fetus. For this reason, some have advocated for a more direct approach attempting to eliminate excessive testing and anxiety. Direct assessment of fetal blood type can be done via invasive prenatal testing. Amniocentesis can allow for the determination of the fetal genotype. Because genotype may not correlate with the RBC antigen expression in some rare instances, direct fetal blood sampling can also be used to determine the fetal phenotype. However, this technique is associated with a higher complication rate and a greater risk of fetal demise as compared to amniocentesis. Recently, newer noninvasive methods to determine fetal genotype have become available. As early as 1997, it was recognized that cell-free fetal DNA could be detected in the maternal circulation, with reports indicating that it was present in detectable quantities as early as 38 days’ gestation. Throughout the years, several companies developed different techniques to identify different loci on the fetal RhD gene. This test became commercially available in Europe and North America. Reports from several European centers specializing in the treatment of RhD disease started to emerge indicating the high accuracy of this test. A meta-analysis looking at over 30 articles concluded that the overall accuracy of this test was around 94% with a sensitivity of 95.4% and a specificity of 98.6%. Several European countries, with different patient populations and insurance payment plans, have started to adopt noninvasive RhD testing as part of the management protocol of women at risk for RhD alloimmunization with success. More worldwide widespread use was limited by the concerns of the lack of data regarding cost analysis. A recent cost analysis done in the United States by Hawk and associates concluded that with the current cost of RhD typing, routine antenatal anti-D immunoglobulin prophylaxis was still less costly. It is possible that with more automated techniques and increased competition driven by higher demand, the cost of testing may decrease, leading to the expansion of the acceptance of this test as part of the RhD alloimmunization management protocol. However, some have raised concern that, because cell-free DNA screening does not detect 100% of RhD-positive fetuses, routine use of cell-free DNA to target RhD immune globulin for RhD-negative women would cause increased sensitization compared with routine RhD immune globulin administration. Assays for detecting RhCc, RhE, and Kell typing via cell-free fetal DNA are also currently available.

Prediction of Anemia

Once a patient has been sensitized, there is a risk for fetal anemia. Several invasive techniques allow for the direct evaluation of fetal hemoglobin and hematocrit. Normally, fetal hemoglobin concentration increases with advancing gestation, and reference ranges for fetal hemoglobin have been established using fetal blood sampling ( Table 17-2 ). Cordocentesis can accurately detect fetal anemia; however, this technique carries with it a risk of pregnancy loss and fetal demise reported to range from 0.9% to 3.2%. Historically, another method used in the prediction of fetal anemia was through amniocentesis. Levels of bilirubin within the amniotic fluid were measured using the ΔOD ₄₅₀ assay to predict hemolysis. The values obtained would be later plotted against published nomograms to determine the risk of severe fetal anemia This approach had the downside that in many cases serial amniotic fluid samplings were required to assess the severity and progression of the disease. These two procedures have been relegated to the past by the widespread use of sonographic Doppler evaluation of the middle cerebral artery (MCA).

Advances in ultrasound technology have led to this technique now being the preferred method of prediction of fetal anemia. As mild to moderate anemia develops, the fetus responds with several compensatory mechanisms. The increased demand is thought to lead to increased ventricular output and stroke volume. These changes, along with a decrease in fetal blood viscosity secondary to the loss of RBCs, lead to an increase in flow velocity. Several fetal vessels were interrogated in an attempt to identify an easily accessible window for evaluation of blood flow. Measurements of the peak systolic velocity (PSV) in the MCA have been found to be highly predictive of severe fetal anemia. This vessel has the benefit of being easily identifiable with low intra­observer and interobserver variability. Because this measurement varies across gestational ages, the Collaborative Group for Doppler Assessment of the Blood Velocity in Anemic Fetuses has developed a reference in which the PSV measurement obtained is converted to multiples of the median (MoM) and used to predict the probability of fetal anemia ( Table 17-3 ). Using a hemoglobin concentration of 5 g/dL as a threshold, a cutoff of 1.5 MoM has been found to be highly predictive of severe fetal anemia requiring therapy in RBC alloimmunization affected pregnancies ( Figs. 17-10 and 17-11 ). This approach led to a false positive rate of approximately 12%. Later studies comparing the accuracy of this measurement to serial amniocentesis determined that this sonographic measurement has a better sensitivity and specificity. With the increasing experience and standardization in obtaining this measurement as well as the improvement in ultrasound technology, measurement of PSV in the MCA has become the surveillance modality of choice in alloimmunized pregnancies.

Nomogram for mild anemia (1.29 MoM) and moderate-severe anemia (1.5 MoM). MCA, middle cerebral artery; MoM, multiples of median; zone A values, mild anemia; zone B values, moderate to severe anemia.

The MCA can be easily identified at the base of fetal skull. Color Doppler is utilized to identify the entire circle of Willis ( Figs. 17-12 and 17-13 ). The examiner should interrogate the MCA closest to the transducer. If this is not possible, evaluation of the MCA in the far field may provide reliable results. The 1- to 2-mm Doppler gate should then be placed just distal to the origin of the MCA from the carotid siphon. The ultrasound probe should be manipulated so a 0-degree angle of insonation is obtained. One approach to help achieve this angle is to start by obtaining the view for a biparietal diameter measurement. The probe should then be angled slightly toward the fetal skull and moved sideways in order to “tilt” the fetal head on the ultrasound screen. This will allow the MCA to be viewed in an almost completely vertical position. Angle correction can sometimes be used if the 0-degree angle insonation cannot be obtained effectively, although this approach is not preferred. Several measurements of the MCA PSV should be obtained, as these can be distorted by fetal movement or breathing. All measurements should have similar values to one another. The best measurement obtained should be used; the values obtained should not be averaged.

Anterior and middle cerebral artery ( arrow ) using color flow Doppler sonography for localization.

(Courtesy of Anthony Swartz, BS, RT(R), RDMS.)

Middle cerebral artery (MCA) assessment at 19 weeks’ gestation. Top image illustrates power Doppler localization of the proximal MCA vessel. Note Doppler gate location. Peak systolic velocity on lower image was measured at 52.42 cm/second; 1.87 MoM for this gestational age. FHR, fetal heart rate; MoM, multiples of the median.

(Courtesy of Anthony Swartz, BS, RT(R), RDMS.)

MCA PSV can be obtained as early as 16 to 18 weeks’ gestation. A multicenter prospective trial has shown that this measurement has a high false positive rate when measured after 35 weeks’ gestational age. Measurements should be repeated every 1 to 2 weeks depending on the clinical presentation. If MCA PSV measurements reach a value greater than 1.5 MoM, fetal blood sampling via cordocentesis should be performed. Should the fetal hematocrit be found to be below 30%, intrauterine transfusion is indicated. Many experts recommend that if the serial evaluation of MCA PSV remains below the established threshold, monitoring should be continued, with a plan for induction of labor at 37 to 38 weeks. Delivery is also warranted if there is any evidence of fetal compromise. Antenatal testing via nonstress test or biophysical profile should also be instituted starting at 32 weeks’ gestation.

Elevated MCA PSV after 35 weeks represents a therapeutic challenge. In the past, some investigators advocated performing amniocentesis to obtain both levels of bilirubin within the amniotic fluid measured using the ΔOD ₄₅₀ assay as well as testing fetal lung maturity. However, the lack of familiarity with performing fetal lung maturity assays within several referral centers added to the fact that there has been a move toward abandonment of fetal lung maturity testing in well-dated pregnancies has led to the abandonment of the practice of amniocentesis beyond 35 weeks for this indication.

For women with a prior history of an affected fetus or child, defined as a fetus requiring intrauterine transfusion or exchange transfusions after birth, the use of serial maternal titers is no longer indicated because they are no longer predictive after an affected pregnancy. Fetal RhD status can be established; if the fetus is RhD positive, monitoring with serial MCA Doppler tests should commence around 16 to 18 weeks. Figure 17-14 summarize a proposed protocol for evaluation and management of affected pregnancies.

Algorithm for the clinical management of the red blood cell alloimmunized pregnancy. GA, gestational age; MCA, middle cerebral artery; MoM, multiples of the median; PSV, peak systolic velocity.

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