Williams Hematology



Definition and History
Etiology and Pathogenesis

Causative Antibodies

RH Hemolytic Disease

ABO Hemolytic Disease

Hemolytic Disease Due to Other Red Cell Antibodies
Clinical Features



Other Clinical Features

Obstetric History
Laboratory Features



Differential Diagnosis
Therapy, Course, and Prognosis

Chapter References

Hemolytic disease of the newborn occurs as a result of sensitization of the mother’s immune system to red cell antigens of the fetus. This sensitization results in the transplacental passage of maternal IgG antibodies that bind to the fetal red cells, causing hemolysis, and as a consequence of the hemolytic process, anemia, extramedullary hematopoiesis, and neonatal hyperbilirubinemia, sometimes with devastating morbidity for the fetus and newborn infant. This chapter discusses the pathophysiology, the recent developments in diagnostic methods, and the preventive and therapeutic strategies that have contributed to a dramatic decrease in the incidence and severity of hemolytic disease of the newborn.

Acronyms and abbreviations that appear in this chapter include: DAT, direct antiglobulin test; DOD450, change in optical density at 450 nm; IgG, immunoglobulin G; IVT, intravascular fetal transfusion.

Alloimmune hemolytic disease of the newborn is a disorder in which the life span of fetal and/or neonatal red cells is shortened due to the binding of transplacentally transferred maternal IgG antibodies on fetal red cell antigens foreign to the mother, inherited by the fetus from the father.
Although the condition was described in newborn infants as early as the 1600s, it was not until 1932 that Diamond, Blackfan, and Baty recognized that the clinical syndromes of stillbirth with unusual erythroblastic activity in the extramedullary sites and blood, fetal hydrops, anemia in the newborn, and “icterus gravis neonatorum” were closely related and were probably due to the same underlying disturbance of the hematopoietic system.1 The discovery of the Rh factor by Landsteiner and Weiner2 in 1940 led to further elucidation of the condition by Levine,3 who established that erythroblastosis fetalis was caused by the red blood cells from immunization of an Rh-negative mother by an Rh-positive fetus. Antibodies produced by the sensitized mother crossed the placenta and coated the fetal Rh-positive cells, leading to hemolysis and thus to anemia, hydrops, and severe neonatal jaundice secondary to hemolysis. Over the next decade, neonatal mortality from Rh hemolytic disease of the newborn was reduced considerably by exchange transfusion techniques for correction of severe anemia and prevention of the extreme hyperbilirubinemia.4 However, severely affected infants continued to die in utero before 34 weeks’ gestation. In 1961, Liley demonstrated the prognostic value of amniotic fluid spectrophotometry to identify these infants and then showed that intrauterine transfusions could prevent fetal deaths.5 The most dramatic reduction in the incidence of Rh hemolytic disease of the newborn was achieved in the sixties and seventies with the development of postpartum and antepartum anti-D prophylaxis to prevent Rh sensitization.6 Progress in the diagnosis and management of both the fetus and the affected newborn infant and prevention of Rh hemolytic disease of the newborn has resulted in a hundred-fold drop in deaths due to Rh hemolytic disease of the newborn in the past century.7 However, the disease has not disappeared, and cases of hemolytic disease of the newborn due to red cell antibodies directed toward antigens other than the Rh blood group system are being increasingly recognized.8,9 and 10
When considering the specificity of alloantibodies that can cause maternal isoimmunization, it is useful to group them into the following three categories: (1) antibodies directed against the D antigen in the Rh blood group system, (2) antibodies directed against the A and B antigens, and (3) antibodies directed against the remaining red cell antigens. Hemolytic disease of the newborn due to the various antibody systems may differ among races, resulting in differences in clinical severity and outcome.
The D antigen in the Rh blood group system is the most important of the three pairs of Rh antigens Cc, Dd, and Ee. Every individual inherits a set of each of the three pairs of antigens from each parent. The presence or absence of D determines the Rh-positive or Rh-negative status of the individual. A mutation deleting the D locus is responsible for the d- or Rh-negative phenotype. Many D-positive individuals are homozygous for D (DD), having inherited the D antigen from both parents. Other D-positive individuals are heterozygous for D (Dd), having inherited a D-containing set from one parent and a non-D-containing set of Rh antigens from the other parent. Therefore, it is evident that all the offspring of a homozygous Rh-positive (DD) man and an Rh-negative (dd) woman will be Rh- or D-positive (Dd), whereas a fetus produced by a heterozygous Rh-positive (Dd) father with an Rh-negative mother (dd) could be either Rh-positive (Dd) or Rh-negative (dd). The probable Rh genotype of the father may be deduced from phenotyping studies, based on gene frequencies in various populations.11 There is considerable racial variability in the prevalence of Rh-negativity. About 15 percent of Caucasians are Rh negative,11 compared to 7 to 8 percent of American blacks, 5 percent of Asian Indians,12 and 0.3 percent of the Chinese.13
Since the mandatory institution of Rh-matched blood transfusions, isoimmunization of Rh-negative women by Rh-positive transfusions is now rare. The potential for immunization of the mother is determined by the existence of maternal-fetal blood group incompatibility and by the extent of feto-maternal hemorrhage. Asymptomatic transplacental passage of fetal red cells occurs in 75 percent of pregnant women at some time during pregnancy or during labor and delivery.14 The incidence of fetomaternal transfusion increases with advancing gestation: from 3 percent in the first trimester, 12 percent in the second trimester, and 45 percent in the third trimester to 64 percent after delivery. The volume of fetal red cells that enters the maternal circulation also increases as pregnancy progresses. The average volume of fetal blood in the maternal circulation following delivery is about 0.1 ml in most women and less than 1 ml in 96 percent of women,15 but intrapartum fetomaternal hemorrhage of more than 30 ml may occur in up to 1 percent of pregnancies.16 Fetomaternal transfusion can result from obstetric procedures such as chorionic villus sampling,17 amniocentesis,18 funipuncture,19 therapeutic abortion, cesarean section and manual removal of the placenta,20 and pathologic conditions such as abdominal trauma, spontaneous abortion, or ectopic pregnancy.
The presence of D-positive red cells in the D-negative mother initially provokes a primary immune response that is weak and slow and consists of IgM antibodies that do not cross the placenta. Subsequently, anti-D IgG antibodies capable of crossing the placenta are produced. In the absence of Rh immunoglobulin prophylaxis, sensitization occurs in 7 to 16 percent of women at risk within 6 months after delivery of the first Rh-positive ABO-compatible fetus and in 2 percent after delivery of an ABO-incompatible fetus.21 Fetomaternal ABO incompatibility offers some protection against primary Rh immunization because incompatible fetal red cells are destroyed rapidly by maternal anti-A and anti-B antibodies, reducing the maternal exposure to Rh D antigenic sites. Repeated exposure to Rh-positive fetal red blood cells, as in a second Rh-positive pregnancy in a sensitized Rh-negative woman, produces a secondary immune response that is marked by the rapid production of large amounts of anti-D IgG antibody. ABO incompatibility confers no protection against the secondary immune response once sensitization has occurred.22 The volume of blood required to cause sensitization is often minuscule. Primary sensitization has been reported in 80 percent of individuals injected with 0.5 ml of Rh-positive cells; secondary immune responses may occur with as little as 0.03 ml of Rh-positive cells. Repetitive exposure to D-positive cells in D-negative intravenous-drug–abusing women who share needles with Rh- positive partners has lead to severe Rh sensitization.23
The reason why most women at risk for development of anti-D do not appear to be sensitized is unclear. Several theories proposed include active T-cell suppression, tolerance induction by small amounts of antigen, and the possibility that low-titer anti-D may not be detected by current diagnostic methods.
The binding of transplacentally transferred maternal anti-D IgG antibodies to D-antigen sites on the fetal red cell membrane is followed by adherence of the coated red cells to the Fc receptors of macrophages with rosette formation, leading to extravascular noncomplement-mediated phagocytosis and lysis, predominantly in the spleen.24 Although Rh antigens are found on fetal cells as early as the seventh week of gestation, the active transport of IgG across the placenta is slow until 24 weeks of gestation. The degree of hemolysis may be influenced by the functional immaturity of the fetal reticuloendothelial system prior to 20 weeks of gestation, maternal IgG levels, the IgG subclass, and the rate of transplacental transfer.25 Antibodies of the IgG1 and IgG3 subclasses, often produced in Rh alloimmunization, have a high affinity for Fcg receptors and are associated with severe disease, while maternal antibodies with specificity for allogeneic monocytes, which block Fcg receptors on mononuclear phagocytic cells, may result in unexpectedly mild hemolytic disease of the newborn.24
Fetal anemia secondary to hemolysis results in compensatory extramedullary hematopoiesis in the liver, spleen, kidneys, and adrenal glands and is associated with an outpouring of immature nucleated red blood cells into the circulation. Increased fetal plasma erythropoietin levels have been reported with severe fetal anemia.26 The marked increase in erythropoiesis in fetuses with hemolytic anemia is accompanied by a down-modulation of platelet as well as neutrophil production.27 Extensive extramedullary hemopoiesis in the liver and spleen may lead to portal and umbilical venous hypertension. Placental function is diminished as a result of trophoblastic hypertrophy and placental edema. Ascites and pleural effusions develop, probably secondary to portal and umbilical venous hypertension.28 Hypoproteinemia due to liver dysfunction results in generalized edema. When the edema and ascites become extreme (anasarca), the fetus is at risk for inadequate placental oxygen exchange. This situation, known also as hydrops fetalis, is postulated to be secondary to cardiac failure and elevated venous pressures together with increased capillary permeability and impaired lymphatic clearance.29 In the final stages of hydrops fetalis, there is hydrothorax, with compression of the lungs, resulting in pulmonary hypoplasia. Prior to the institution of intrauterine transfusions, most of these infants died in utero or soon after birth.
Although fetal bilirubin levels are elevated secondary to hemolysis,32 the placenta effectively transports most of the lipid-soluble unconjugated fetal bilirubin, so the infant is not clinically jaundiced at birth. In severe cases, bilirubin secreted from the fetal trachea stains the amniotic fluid, umbilical cord, and vernix caseosa.
Some infants with hemolytic disease develop anemia beyond the immediate neonatal period lasting up to 8 to 12 weeks of age. Delayed anemia is related to continuing hemolysis due to persistence of maternal antibodies30 and a hyporegenerative component with decreased red cell production, associated with low serum concentrations of erythropoietin.31
ABO hemolytic disease of the newborn is limited to mothers who are blood group type O and whose babies are group A or B. Although ABO incompatibility exists in 15 percent of O group pregnancies, ABO hemolytic disease is estimated to occur only in about 3 percent of all births. Although far more common than Rh hemolytic disease of the newborn, ABO hemolytic disease of the newborn is usually mild and rarely responsible for fetal deaths. A higher incidence and greater severity is reported in southeast Asians, Latin Americans, Arabs, and South African and American blacks,33,34,35 and 36 but even in these populations the clinical phenomenon is early neonatal jaundice requiring phototherapy or exchange transfusions. Severe fetal anemia and hydrops has been rarely reported (Table 58-1).37,38


There are many reasons for the low incidence and severity of ABO hemolytic disease of the newborn despite considerable fetomaternal ABO incompatibility. Most anti-A and anti-B antibodies are of the IgM type and do not cross the placenta. A small number of group O women produce anti-A and anti-B antibodies of the IgG type that can cross the placenta. The severity of the disease in the infant may relate in part to the level of IgG anti-A or anti-B in the mother and the IgG subclass. IgG2 constitutes a significant component of anti-A and anti-B antibody; this subclass of IgG is transported less readily across the placenta than are IgG1 or IgG3 and is a less efficient mediator of macrophage-induced red cell clearance.11 There are a small number of fully developed A or B antigen sites on fetal red blood cells. IgG anti-A and anti-B are absorbed onto other tissues bearing these surface antigens, thereby diluting their effect. In addition, individuals of the type O blood group have different lymphocyte precursor frequencies, resulting in different titers of anti-A and anti-B, than do individuals of A, B, or AB blood groups.39
Unlike Rh disease, ABO hemolytic disease of the newborn occurs with the same frequency in the first as in subsequent pregnancies, since maternal anti-A and anti-B antibodies are present normally, probably secondary to sensitization against A or B substances in food or bacteria. Anti-A and anti-B IgG antibodies do not bind complement on the fetal red cell membrane40; hemolysis occurs by noncomplement-mediated phagocytosis of Ig-coated red cells, similar to Rh hemolytic disease of the newborn. The blood film in ABO hemolytic disease of the newborn is marked by the presence of microspherocytes, a feature not seen in Rh hemolytic disease of the newborn.41 The spherocytosis is postulated to be due to loss of membrane surface area when the spleen removes antigen-antibody complexes from the affected cell. Increased osmotic fragility and autohemolysis, similar to hereditary spherocytosis, may be demonstrated in ABO hemolytic disease of the newborn, but, unlike hereditary spherocytosis, the autohemolysis in ABO hemolytic disease of the newborn is not corrected by the addition of glucose.
Antenatal screening programs detect clinically significant antibodies in 0.24 to 1 percent of pregnant women.8,9 and 10 Despite the success of Rh prophylaxis, anti-D antibodies still constitute a large proportion of the antibodies detected. When D and ABO are excluded, non-D Rh antibodies (c, C, e, E, cc, and Ce) and those belonging to the Kell, Duffy, Kidd, and MNS systems are most frequently involved (Table 58-2). Although the list of antibodies reported to cause hemolytic disease of the newborn includes IgG specific for virtually any known red cell antigen, some specificities are seen more frequently in severe cases. Anti-c, anti-Kell, and anti-E may cause hemolytic disease of the newborn as severe as that seen in anti-D hemolytic disease of the newborn.42


Kell hemolytic disease accounts for 10 percent of the cases of antibody-mediated severe fetal anemia.43 The Kell blood group system is composed of at least 24 discrete antigens. The Kell antigen (also called KEL1 or K1) is expressed by erythroid progenitor cells and mature erythroid cells, but in only 9 percent of individuals. Alloimmunization in Kell-negative women is often the result of blood transfusion rather than sensitization by fetomaternal hemorrhage from a Kell-positive fetus.44,45 and 46 Kell hemolytic disease is rare in alloimmunized pregnancies because fetal anemia due to transplacentally transmitted antibodies can occur only in a Kell-positive fetus. The partners of Kell-negative women are likely to be Kell positive only in 10 percent of pregnancies, and only half of these pregnancies are likely to be incompatible because of paternal heterozygosity. Published results on the outcome of maternal Kell alloimmunization indicate that between 2.5 and 10 percent of Kell-immunized pregnancies end in the delivery of affected infants,44,47,48 with about half the infants requiring intervention. Unlike anti-D alloimmunized pregnancies, maternal antibody titers and amniotic fluid readings in Kell-alloimmunized pregnancies fail to reflect the severity of the disease in the affected fetus.48 Affected fetuses also have inappropriately low levels of circulating reticulocytes and normoblasts for the degree of anemia, secondary to specific suppression of erythropoiesis at the progenitor cell level by anti-Kell antibodies.49
Anemia, jaundice, and hepatosplenomegaly are the hallmarks of hemolytic disease of the newborn. The clinical spectrum of affected infants is highly variable. In Rh hemolytic disease of the newborn, half of the infants have very mild disease and do not require intervention. One-quarter of affected infants are born at term with moderate anemia and develop severe jaundice. In the days prior to intrauterine intervention, hydrops developed in utero in the remaining one-quarter, with half becoming hydropic prior to 34 weeks’ gestation. In Kell hemolytic disease of the newborn, the clinical spectrum of hemolytic disease of the newborn is less predictable, ranging from limited clinical stigmata to frank hydrops. Anemia, jaundice, and hepatosplenomegaly are also seen in ABO hemolytic disease of the newborn, but the disease is usually milder than is Rh hemolytic disease of the newborn.
Infants with mild hemolytic disease of the newborn have cord blood hemoglobin concentrations slightly lower than the age-related normal range. Hemoglobin values usually begin to fall during the first 24 h of life, and hemolysis continues until all incompatible red cells and/or circulating maternal alloantibody is eliminated from the circulation. Since the alloantibodies are IgG, the half-life is approximately 3 weeks. Physical examination in infants with moderate to severe anemia will reveal pallor, tachypnea, and tachycardia. Signs of cardiovascular collapse and tissue hypoxia appear when anemia is severe (hemoglobin <4 g/dl, hematocrit 15%).
Most infants with hemolytic disease are not jaundiced at birth. In untreated patients with mild disease, the serum-indirect bilirubin peaks by the fourth or fifth day and then declines slowly. Premature infants may have greater levels of serum bilirubin due to lower activity of hepatic glucuronyl transferase activity. The umbilical cord and vernix caseosa may be stained with bilirubin from the amniotic fluid in severely affected infants. Clinical icterus usually develops during the first day of life, often in the first few hours of life, in such infants, progressing in a cephalopedal direction with rising bilirubin levels. Infants who have received intrauterine transfusions may have marked conjugated hyperbilirubinemia at birth.
An important complication of significantly elevated serum levels of indirect bilirubin in the neonate is the development of bilirubin encephalopathy.50 This disorder, also termed kernicterus, is caused by bilirubin pigment deposition, leading to neuronal necrosis in the basal ganglia and cerebellum. Bilirubin encephalopathy is initially marked by lethargy, poor feeding, and hypotonia. With increasing severity, the infant develops a high-pitched cry, fever, hypertonia progressing to frank opisthotonos, and irregular respiration; 50 percent of affected term infants die at this stage. The hypertonia becomes less pronounced in surviving infants, who then develop any or all of the classic sequelae of choreoathetoid cerebral palsy, upward gaze palsy, sensorineural hearing loss, and mental retardation. Presentation in preterm infants is less characteristic, but the mortality is higher. Occasionally, infants may have subclinical bilirubin encephalopathy in the neonatal period, manifesting later with the development of mild motor or cognitive dysfunction.
Infants with hemolytic disease of the newborn, particularly those with alloimmune hemolytic disease of the newborn, are at higher risk for kernicterus than are other infants with the same bilirubin level. There are several possible explanations for this finding. Heme pigments might inhibit bilirubin-albumin binding. Alternatively, the complex in utero physiology of erythroblastosis with acidosis and cerebral hypoxia may compromise the blood-brain barrier. Other factors that predispose to kernicterus include hypothermia, hypoglycemia, sepsis, hemolysis, and prematurity. Many of these conditions are present in severely affected infants.
Hepatosplenomegaly is usually present, and the degree usually correlates with severity of the disease. For those infants who survive, no laboratory evidence of liver disease is evident. The most marked hepatosplenomegaly is seen in infants with hydrops fetalis, who also have peripheral edema and ascites. Respiratory distress may be present due to pulmonary hypoplasia or pleural and/or pericardial effusions or may be due to surfactant deficiency. Purpura associated with thrombocytopenia is commonly seen in severely affected infants and may be a bad prognostic sign. The placenta is thickened, enlarged, and pale.
The course and outcome of prior pregnancies is of paramount importance in the initial evaluation of an alloimmunized pregnancy. The history of early fetal deaths or hydrops is ominous. In Rh alloimmunization, the severity of hemolytic disease of the newborn either remains the same or worsens in subsequent affected pregnancies. Hydrops recurs in 90 percent of affected pregnancies, often at an earlier gestation. Jaundice due to hemolysis is also likely to recur to the same degree of severity in subsequent affected pregnancies. The history of prior blood transfusions is important in sensitization to antibodies other than D, particularly Kell alloimmunization. The establishment of paternity for each pregnancy is particularly relevant in both Rh and Kell alloimmunization, since the fetus is at risk only if the father is positive for the antigen in question. ABO hemolytic disease of the newborn may affect the first-born ABO-incompatible infant. Although rare, severe ABO hemolytic disease of the newborn may also recur in subsequent ABO-incompatible pregnancies.37
The aims of antenatal serological testing are to identify Rh-negative women for whom anti–D immunoglobulin prophylaxis will be required, to identify maternal alloimmunization, and to ascertain the risk to the fetus from alloimmune hemolytic disease. Every obstetric patient should have ABO and Rh-D typing and be tested for irregular serum antibodies, irrespective of Rh type, at the initial prenatal visit, preferably by 12 to 16 weeks’ gestation. Women who initially test as Rh negative should be tested for the weak-D phenotype, also termed as Du or D+w. Tests with enzyme-treated red cells or polyspecific antiglobulin sera are not recommended, since they may detect clinically insignificant antibodies. The current American Association of Blood Banks standards for blood banks and transfusion testing recommend repeat Rh testing only in women undergoing delivery, abortion, or an invasive obstetric procedure, or if there is a request for red blood cell transfusion. Antibody screening is repeated at 28 to 30 weeks’ gestation only in Rh-negative women, while in Britain and Canada all women undergo repeated screening for irregular antibodies regardless of Rh type.51 A weakly reactive anti-D (titer of 4 or less) may be demonstrated in women who have received antenatal Rh immunoglobulin and should not be mistaken for sensitization.52
If the mother is found to be alloimmunized, the specificity of the antibody and its ability to cause hemolytic disease of the newborn need to be determined. Antibody quantification is usually performed by titration using the indirect antiglobulin test, with different laboratories establishing “critical titers” varying from 8 to 32 for Rh-D antibodies. Specimens are frozen, and successive titration is performed using the same methods. The trend in sequential antibody levels, together with the previous obstetric history, is considered more important than any isolated level in predicting disease severity.51,53,55 Serological tests in alloimmunized women may be measured every 2 to 4 weeks from 18 weeks’ gestation, with rapidly rising levels or a critical titer or level dictating further investigation. The significance of titer levels for antibodies other than D have not been defined. Maternal anti-Kell titers, in particular, correlate poorly with fetal outcome.43
The imperfect predictive value of serological tests has led to the development of functional cellular assays that measure the ability of maternal antibodies to cause red cell destruction. In these assays, red blood cells sensitized with maternal antibodies are incubated with effector cells carrying Fcg receptors, such as lymphocytes or monocytes. Cellular interaction, such as binding, phagocytosis, or cytotoxic lysis, is measured by different techniques 24,54,56
The standard 300-µg dose of anti-Rh immunoglobulin (RhIg) affords protection against 30 mL of Rh-positive blood. However, fetomaternal hemorrhage in excess of 30 mL may occur in women without predisposing risk factors.16 All Rh-negative nonimmunized women should have blood tested approximately 1 h after delivery of an Rh-positive baby for fetomaternal hemorrhage. During the antenatal period, testing is indicated after 20 weeks’ gestation if clinical circumstances suggest the possibility of excessive transplacental hemorrhage (e.g., abdominal trauma or abruptio placentae). Both a rosette test57 and an enzyme-linked antiglobulin test58 are recommended methods of screening for excessive fetomaternal hemorrhage at delivery. If the rosette test result is positive, the number of fetal red cells should be determined. The Kleihauer-Betke test,59 the standard test in use in most laboratories, permits quantification of fetal hemoglobin-containing red cells in a maternal blood sample. The test is based on the resistance of fetal hemoglobin, unlike adult hemoglobin, to acid elution. False-positive results may be obtained in conditions that are associated with increased fetal hemoglobin, such as hereditary persistence of fetal hemoglobin, sickle cell disease, or sickle cell trait. Flow cytometric methods appear to offer increased accuracy and reliability,60,61
The child of an Rh-negative mother and a heterozygous Rh-positive father has a 50 percent chance of being Rh negative and thus being unaffected by prior maternal Rh alloimmunization. When the father is heterozygous or when paternal zygosity is unknown, the determination of fetal blood type early in pregnancy allows the early institution of monitoring and therapy in Rh-D–positive fetuses who are at risk and the avoidance of invasive procedures if the fetus is Rh negative. Early diagnosis also allows for termination of pregnancy in women who are unwilling to undergo the frequent invasive procedures often necessary for the salvage of severely affected fetuses. Fetal blood sampling for serologic blood typing is associated with a 40 percent risk of fetomaternal hemorrhage and worsening maternal sensitization, and up to 2 percent risk of fetal loss.19 Rh-D–positive fetal cells may be detected rapidly in chorionic villus samples by flow cytometry.62 Cloning of the human Rh-D gene63 has facilitated fetal Rh-D genotyping from small samples of fetal cells obtained by chorionic villus sampling or amniocentesis by the use of polymerase chain reaction techniques.64 The risks of augmenting maternal sensitization and fetal loss even by these procedures have been eliminated by noninvasive methods of prenatal diagnosis of fetal Rh-D status, through the use of fetal cells isolated from the maternal blood,65,66 and 67 and by using fetal DNA extracted from maternal plasma early in the second trimester of pregnancy.68
Prenatal determination of the Kell genotype, necessary for analyzing the possible risk to the fetus in Kell alloimmunization, may be performed either by flow cytometry,62 by DNA amplification of fetal tissue69 obtained by chorionic villus sampling, or from amniocytes.70
In 1961, Liley reported that the spectrophotometric analysis of amniotic fluid for bilirubin was useful in predicting the severity of fetal anemia from 27 weeks to term. Elevations of optical density at 450 nm (DOD450) reflect the concentration of amniotic fluid bilirubin derived from fetal tracheal and pulmonary secretions. The change in optical density is quantified by measuring the elevation of the optical density at 450 nm above a line connecting the optical density values obtained at 375 and 550 nm, and then plotting it against gestational age. Contamination of amniotic fluid samples with blood or meconium make DOD450 readings impossible. Liley defined three zones, with readings in zone 3, the upper zone, indicating severe fetal disease with hydrops or impending fetal death; zone 1, the lowest zone, indicating mild or no hemolytic disease with a 10 percent risk of needing a postnatal exchange transfusion; and zone 2, indicating moderate disease. Serial determinations of DOD450 can achieve a sensitivity of 95 percent in detecting the severity of fetal anemia in the third trimester of pregnancy42 but are unreliable during the second trimester.71 Modifications of the Liley zones before 25 weeks’ gestation48,72 may help to determine whether fetal blood sampling is indicated for definitive diagnosis and treatment. Ultrasound-guided amniocentesis carries a 2.5 percent risk of fetomaternal hemorrhage with the possibility of worsening alloimmunization.18
Ultrasonography is noninvasive, can be performed serially, and may be combined with other diagnostic studies to assess the fetal condition, to estimate the need for further aggressive management, and to obtain a biophysical profile of the fetus to determine fetal well-being. Hepatosplenomegaly, ascites, edema, or frank hydrops can be detected. The earliest ultrasound signs of cardiac decompensation are a small pericardial effusion and dilatation of the cardiac chambers. In the absence of hydrops, ultrasonographic parameters such as intra- and extrahepatic vein diameters, abdominal and head circumference, head–abdominal circumference ratio, and intraperitoneal volume have been unreliable in distinguishing mild from severe fetal anemia.73 Fetal splenic circumference is a sensitive indicator of severe anemia in nonhydropic cases without prior transfusion.74 Preliminary data show that Doppler monitoring of flow velocity indices in the middle cerebral artery and umbilical vein may also be useful noninvasive techniques.75,76
Percutaneous umbilical blood sampling allows for direct measurement of blood indices to specifically evaluate the degree of severity of fetal hemolytic disease as early as 17 to 18 weeks’ gestation.77 Specimens of fetal blood are obtained for direct measurement of complete blood count, reticulocyte count, red cell antigen phenotyping, direct antiglobulin test, bilirubin, blood gases, and lactate to assess acid-base status. To exclude maternal blood contamination, fetal blood should be examined using a number of fetal-specific markers, such as red cell size, hemoglobin F, and/or expression of the i red-cell antigen.78 Indications for perimumblical blood sampling in alloimmunized pregnancies include fetal Rh typing, DOD450 measurement in Liley zone III or rising through zone II, when an anterior placenta precludes amniocentesis in a fetus where maternal history or antibody titers indicate high fetal risk, or ultrasonographic evidence of early or frank hydrops.79 For a woman with a previous alloimmunized pregnancy, umbilical blood sampling with transfusion should be timed 10 weeks before the time of the earliest previous fetal or neonatal death, fetal transfusion, or birth of a severely affected baby, but not before 18 weeks’ gestation unless hydrops is evident. The use of management protocols can reduce the need for multiple invasive procedures while providing specific information about fetal status.80 Fetal blood samples with reticulocyte counts greater than the 97.5 percentile for gestation, a strongly positive direct Coombs test result, or a mild anemia (hematocrit >30% but <2.5 percentile for gestation) predict fetuses at high risk of having significant antenatal anemia, thus requiring frequent ultrasonographic monitoring and repeated cordocentesis at 1- to 2-week intervals to determine whether intrauterine transfusion is warranted.81 Complications of fetal blood sampling include fetal loss, with procedure-related rates ranging from 0 to 4.9 percent, umbilical cord bleeding, fetal bradycardia, chorioamnionitis, and a significant risk of fetomaternal hemorrhage with anamnestic maternal sensitization.82
A sample of cord blood should be collected at the time of delivery from all newborns. However, specific testing of cord blood samples is performed only if the mother is Rh negative, if when the maternal serum contains red cell alloantibodies of potential clinical significance, or if the neonate develops signs of hemolytic disease. Tests should include ABO and Rh typing and a direct antiglobulin test (DAT). Occasionally, high titers of maternal antibody may block Rh-antigenic sites on the neonatal red cells, leading to false-negative Rh typing.
Antepartum RhIG given to the mother may result in a weakly positive DAT result in the infant at birth.52 Contamination of the cord blood sample with Wharton’s jelly during collection can result in a false-positive DAT result. Although the antiglobulin test usually is positive in all forms of hemolytic disease of the newborn, it cannot predict reliably the degree of clinical severity. This is especially true for cases due to ABO sensitization. Elution of maternal antibody from the infant’s red cells, followed by tests to determine the specificity of the antibody in the eluate may be useful, particularly when several antibodies are present in the maternal serum.
Cord blood hemoglobin and indirect bilirubin determinations more closely reflect disease severity. Most infants with cord hemoglobin levels within the age-adjusted normal range do not require exchange transfusion. In these infants, determination of cord-indirect bilirubin is more valuable. Usually, a cord hemoglobin level of less than 11 g/dl and/or a cord-indirect bilirubin level of greater than 4.5 to 5 mg/dl warrants exchange transfusion. Early exchange transfusion may also be indicated if the rate of rise of bilirubin, measured every 4 to 6 h, exceeds 0.5 mg/dl/h.
The reticulocyte count is usually more than 6 percent and may approach 30 to 40 percent in severe Rh disease.41 The peripheral blood smear is characterized by increased nucleated red blood cell counts, polychromasia, and anisocytosis. Severely affected infants may develop thrombocytopenia with platelet counts below 30,000/ml. Spherocytosis is usually seen only in ABO hemolytic disease. Low reticulocyte counts disproportionate to the low hematocrit are evident in Kell hemolytic disease of the newborn.
Hypoglycemia, secondary to hyperinsulinemia, is also seen in severely affected infants. Arterial blood gas analysis may reveal metabolic acidosis and/or respiratory decompensation. Hypoalbuminemia is often present.
Cardiomegaly and pleural and pericardial effusions may be evident on radiological investigation. Cardiac hypertrophy with disproportionate septal hypertrophy has been noted in severely affected infants by echocardiography.83
Infants who have received intrauterine transfusions may have mild or moderate anemia with little reticulocytosis. Since most of their circulating red cells are transfused antigen-negative cells, the direct antiglobulin test result may be negative, but the indirect antiglobulin test result will be strongly positive.
Hydrops fetalis may be secondary to cardiac anomalies or arrhythmias, fetal genetic or metabolic disorders, intrauterine infections such as syphilis or toxoplasmosis, or any of a multitude of causes that lead to severe derangements in fetal homeostasis. These disorders may be classified as nonimmune hydrops and are differentiated from anasarca secondary to hemolytic disease of the newborn by the absence of any clinically significant red cell alloantibodies in the mother’s blood. Parvovirus B19 infection of the mother at any point in gestation can cause nonimmune hydrops, profound fetal anemia, and death.
Neonatal anemia due to intrinsic red cell defects such as hereditary spherocytosis, red cell enzyme deficiencies, and specific hemoglobinopathies can give a similar clinical picture to hemolytic disease of the newborn. The absence of maternal red cell alloantibodies, a negative direct antiglobulin test result, and the detection of the specific defect determining the disorder will clarify the diagnosis.
Disorders of bilirubin metabolism, either indirect, direct, or a combination of both pigments, usually are not associated with anemia. Also, hepatitis or obstructive biliary diseases may present with hyperbilirubinemia. The direct antiglobulin test result is negative except in those cases that happen to be from ABO-incompatible pregnancies. In these instances, a positive direct antiglobulin test result usually does not reflect associated hemolytic disease of the newborn.
Intrauterine Fetal Transfusion Intraperitoneal fetal transfusion has been largely replaced by direct intravascular fetal transfusion (IVT) by funipuncture. Other techniques of fetal transfusion reported include intrahepatic venous puncture, combinations of intravascular with intraperitoneal transfusions, and even intracardiac transfusion as a last resort.84 Intraperitoneal fetal transfusion was performed when serial amniotic fluid spectrophotometric measurements rose into upper zone II before 30 weeks’ gestation or into zone III before 32 to 34 weeks’ gestation. Intraperitoneal transfusions may be necessary when intravascular access is difficult, as in early pregnancy when the umbilical vessels are narrow or later when increased fetal size prevents access to the umbilical cord. The intravascular technique offers precise diagnostic evaluation of the fetal status (see Percutaneous Umbilical Blood Sampling, above) and is effective even in hydropic fetuses by circumventing the problem of erratic and often poor absorption of red blood cells from the peritoneal cavity in such fetuses. The relative merits of direct simple intravascular transfusion versus intravascular exchange transfusion have been debated, but the shorter procedure time with direct simple IVT has made it the procedure of choice at most centers.
The first umbilical blood sampling with transfusion ideally should be performed when the fetus is anemic but before hydrops has developed. Transfusions are performed at hematocrit levels of 25 to 30 percent or less. Generally, the hematocrit drops by 1 to 2 percent per day in the transfused hydropic fetus; the fall in hematocrit is rapid in fetuses with severe hemolytic disease, necessitating a second transfusion within 7 to 14 days; the interval between subsequent transfusions is usually 21 to 28 days. Very low pretransfusion fetal hematocrit levels, rapid large increases in posttransfusion hematocrit level, and increases in umbilical venous pressure during IVT are associated with fetal death post-transfusion.85,86
Freshly packed O-negative red blood cells that are antigen negative for any other identified antibody, cytomegalovirus seronegative or leukodepleted, irradiated, and cross-matched against the mother’s blood are used.87 Fetuses are at risk for both posttransfusion cytomegalovirus and graft-versus-host disease. Blood that is washed free of the anticoagulant citrate and other additives and that has maximal in vivo survival is advocated. Acidosis is to be avoided so that the hemoglobin oxygen affinity is not reduced due to a shift in the hemoglobin dissociation curve. Blood is prepared to increase the fetal hematocrit to between 40 and 45 percent. The blood should be as fresh as possible, warmed, and packed to a hematocrit of 70 to 85 percent in a volume calculated based on estimated fetal placental blood volume, fetal hematocrit, and hematocrit of donor blood.
Delivery The decision as to when to deliver the fetus is based on gestational age, fetal weight and lung maturity, fetal response to the transfusions, and the ease of performing the transfusion combined with the antenatal ultrasound and Doppler studies. Transfusions are provided up to 33 to 34 weeks, with delivery as soon as lung maturity is achieved by antenatal steroid therapy. Less severely affected fetuses may be allowed to proceed to term before delivery.
Immunomodulation Other treatments used in Rh-sensitized pregnancies include intravenous IgG, plasmapheresis, plasmapheresis combined with intravenous immunoglobulin, glucocorticoids, oral enteric-coated D-positive erythrocytes, and promethazine hydrochloride.88 Each of these modalities attempts a different kind of immune modulation or suppression to reduce antibody response to antigen and may have an adjunctive role in the treatment of severe isoimmunization.89
Results of antenatal monitoring and obstetric interventions during pregnancy, together with the history of the outcome of previous pregnancies, allows the neonatal team to anticipate the needs of the infant born with hemolytic disease. In infants with severe hemolytic disease, severe anemia and hydrops are the immediate life-threatening concerns and are often accompanied by perinatal asphyxia, surfactant deficiency, hypoglycemia, acidosis, and thrombocytopenia. The prevention of kernicterus and neurotoxicity due to severe unconjugated hyperbilirubinemia is the next pressing problem. Phototherapy and exchange transfusions are the main treatment modalities.
The resuscitation and stabilization of hydropic infants is often difficult and involves prompt intubation and positive-pressure ventilation with oxygen. Drainage of pleural effusions and ascites may be required to facilitate gas exchange. Metabolic acidosis and hypoglycemia should be corrected. A partial exchange transfusion may be performed using packed red cells to improve hemoglobin levels and oxygenation; a double-volume exchange transfusion is contemplated only after the initial stabilization.
Infants who have received multiple intrauterine transfusions are delivered closer to term and often require less phototherapy and fewer exchange transfusions in the neonatal period.90,91 However, some still have significant hemolytic anemia at birth, requiring aggressive intervention, and many require additional simple transfusions for severe and prolonged hyporegenerative anemia secondary to suppression of fetal erythropiesis.92
Exchange Transfusion Exchange transfusion removes sensitized red blood cells, bilirubin, and free maternal antibody in the plasma; corrects anemia; and, when a double blood volume exchange is performed (calculated as 2 × 80 ml/kg), replaces 90 percent of the infant’s blood volume with antigen-negative red blood cells that should have normal in vivo survival.
The indications for early exchange transfusions, performed within 9 to 12 h of birth, although debated, have remained essentially unchanged over the last 40 years, with minor modifications. Cord hemoglobin levels £110 g/liter, cord bilirubin levels ³5.5 mg/dl, and rapidly rising bilirubin levels ³0.5 mg/dL/hr despite phototherapy are commonly used criteria for early exchange transfusions ³0. “Late” exchange transfusions are performed when serum bilirubin levels threaten to exceed 20 mg/liter in term infants, the level at which the risk of kernicterus is approximately 10 percent. Exchange transfusions are performed at lower bilirubin levels in premature infants, particularly those with hypoxemia, acidosis, and hypothermia (see Table 58-2). Conjugated or direct bilirubin values are not subtracted from total bilirubin levels when considering levels for exchange transfusions, unless the direct reacting portion exceeds 50 percent of the total bilirubin.
A double volume exchange should eliminate more than 50 percent of the intravascular bilirubin removed. However, the amount of bilirubin is often less, reflecting the equilibrating tissue-bound pool. The use of albumin prior to exchange transfusion in an effort to mobilize tissue bilirubin is controversial. Equilibration of extravascular and intravascular bilirubin and continued breakdown of sensitized and newly formed red cells by persisting maternal antibodies result in a rebound of bilirubin following initial exchange transfusion, often necessitating repeated exchange transfusions in severe hemolytic disease. In infants with ABO hemolytic disease, single-volume exchange transfusions have been shown to be comparable to double volume exchange transfusions.
Blood chosen for the exchange should be ABO compatible, Rh negative, negative for the antigen responsible for the hemolytic disease, and cross-matched against the mother’s blood. Irradiated citrate-phosphate-dextrose blood is prepared as whole blood or reconstituted whole blood (red cells suspended in saline solution, albumin, or plasma) with a hematocrit of 40 to 50 percent warmed through a temperature-controlled in-line blood warmer.93 Hypoxemic or acidotic infants should receive blood known to lack hemoglobin S. Additive solution anticoagulants are avoided, but the blood should be as fresh as possible (<7 days) to maximize the in vivo survival of the transfused red cells.
Exchange transfusions may be performed by the traditional push-pull method with a single vascular access, usually the umbilical vein, or by isovolumetric techniques utilizing two access sites for simultaneous removal of the infant’s blood and administration of new blood.94 Aliquots of 5 to 20 ml, with a maximum of 5 ml/kg, are withdrawn or infused in the discontinuous method at a rate not exceeding 5 ml/kg every 3 min to avoid rapid fluctuations in arterial pressure, which are accompanied by changes in intracranial pressure. When an isovolumetric exchange is being done, volumes to be removed or reinfused should not exceed 2 ml/kg/min. The duration of the exchange is usually 1 to 2 h.
Potential complications of exchange transfusion include hypocalcemia, hyper- or hypoglycemia, thrombocytopenia, dilutional coagulopathy, neutropenia, disseminated intravascular coagulation, umbilical venous and/or arterial thrombosis, necrotizing enterocolitis, and infection. Despite advances in the management of critically ill newborn infants, morbidity and mortality associated with exchange transfusions remains high, particularly in infants who are premature or sick or both. The risk of death or permanent serious sequelae has been estimated to be as high as 12 percent in sick infants, compared with less than 1 percent in healthy infants in a recent study.95 Careful clinical judgment is required in balancing the potential risk of adverse events from exchange transfusion with the risk of bilirubin encephalopathy in ill infants.
Phototherapy The exposure of bilirubin to light results in structural and configurational isomerization and photo-oxidation of bilirubin to less toxic and less lipophilic products that are excreted efficiently without hepatic conjugation. Phototherapy is the prime treatment for unconjugated hyperbilirubinemia, with the aim of treatment being prevention of bilirubin neurotoxicity.
Intensive phototherapy has been found to effectively reduce bilirubin levels and decrease the need for exchange transfusions for hyperbilirubinemia in ABO and Rh hemolytic disease of the newborn.96,97 and 98 Earlier protocols called for the early institution of phototherapy in all infants with hemolytic disease, resulting in the unnecessary, albeit usually benign, treatment of large numbers of infants with mild hemolytic disease whose bilirubin levels would not have risen to nonphysiologic levels even without treatment. Early and intensive phototherapy should be initiated in infants with moderate or severe hemolysis or in infants with rapidly rising bilirubin levels (>0.5 mg/dl/h). Phototherapy is indicated at lower levels for preterm or sick infants (Table 58-3). The effectiveness of phototherapy may be influenced by the wavelength and irradiance of light, the surface area of exposed skin, and the duration of exposure.


Other Treatments Preliminary studies with high-dose intravenous immunoglobulin have shown reduced bilirubin levels and decreased need for exchange transfusions in infants with hemolytic disease.99,100 The decrease in bilirubin levels in IVIG-treated infants is attributed to reduction in hemolysis, probably secondary to blockade of reticuloendothelial Fc receptors. There is increasing interest in the use of synthetic heme analogs. By competitively inhibiting the activity of heme oxygenase, the rate-limiting enzyme in the catabolism of heme to biliverdin, such analogs can suppress bilirubin production. Sn-protoporphyrin, a potent heme oxygenase inhibitor, has been shown to blunt the postnatal rise and peak bilirubin levels in term newborns with ABO hemolytic disease.101 Further documentation of safety and effectiveness of these treatment modalities will be required before they are widely used. Recombinant human erythropoietin decreases the need for postnatal transfusions in infants with late hyporegenerative anemia of Rh hemolytic disease.102
The use of RhIg has dramatically decreased the incidence of hemolytic disease of the fetus and newborn. The mechanism by which RhIg prevents sensitization to the D antigen is not understood. One of the theories proposed is that passively administered anti-D attaches to the D-antigen sites on Rh-positive red blood cells in the circulation and interferes with the host’s primary immune response to the foreign antigen. RhIg also may inhibit antigen-induced B-cell responsiveness by stimulating an increase in suppressor T cells. The postpartum administration of RhIg to all nonsensitized Rh-negative women who deliver an Rh-positive infant decreases the incidence of Rh isoimmunization from 12 to 13 percent to approximately 2 percent. However about 1.8 percent of Rh-negative women are apparently sensitized during pregnancy from small asymptomatic transplacental hemorrhages. Further reduction in the incidence of Rh-isoimmunization to 0.1 percent has been achieved by antepartum RhIg prophylaxis at 28 to 30 weeks’ gestation.52 Although the cost-effectiveness of routine antepartum prophylaxis is questioned, it has been recommended in the United States since 1981 and was recently endorsed in the United Kingdom.103 The standard dose in the United States, 300 µg RhIg (1500 IU), affords protection against a fetomaternal transfusion of 15 ml of Rh-positive red blood cells or 30 ml of Rh-positive whole blood. Recommendations for the routine prophylactic dose vary around the world.104,105 Table 58-4 indicates the recommended dosage of RhIg for prevention of sensitization in the United States.52 Testing with the Kleihauer-Betke test is recommended as a routine in the postpartum period and antenatally if clinical circumstances suggest the possibility of excessive fetomaternal hemorrhage, to determine if additional doses of RhIg are indicated. The failure to implement current recommendations is estimated to be responsible for almost 40 percent of recent cases of Rh isoimmunization.9,105 Despite appropriate Rh prophylaxis, about 0.1 percent of Rh-negative women may be sensitized prior to 28 weeks’ gestation.


Monoclonal anti-RhIg, currently in phase I trials,106 may replace polyclonal RhIg derived from human plasma from immunized volunteer donors in the future.
Prophylaxis similar to RhIg does not yet exist for alloimmunization to antigens other than D. Transfusion of blood compatible with not only the D antigen but also Kell and other Rh antigens has been advocated for premenopausal women to prevent alloimmunization.8,9
In Manitoba, Canada, perinatal mortality from hemolytic disease dropped from 100 per year in the 1940s in a population of 1 million to 1 every 3 years in the mid 1990s.7 Similar reductions have been described in the United States and United Kingdom. There is little doubt that Rh immunoprophylaxis played a critical role in the decline of perinatal mortality due to Rh hemolytic disease. Changes in birth-order distribution and improvements in the quality of perinatal care have also been important factors.107 Prior to the development of treatment measures in the 1940s, almost half of all newborn infants with Rh hemolytic disease died or were severely handicapped. Perinatal survival rates of over 90 percent have been achieved with intrauterine transfusions in nonhydropic fetuses with severe Rh hemolytic disease.84 The survival rate for hydropic fetuses is lower, at 74 percent, despite intrauterine transfusions, but still remarkable considering nearly all would have perished in the 1960s.
The neurodevelopmental outcome for infants saved by intrauterine transfusion has generally been excellent, with more than 90 percent of survivors being free of disability.90,108 Perinatal asphyxia and lower cord hemoglobin level at birth have been associated with an increased risk of neurologic abnormalities. Neurological abnormality due to extreme indirect hyperbilirubinemia secondary to alloimmune hemolytic disease has virtually disappeared in the United States and Canada but is still seen in countries with more limited resources.109

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Wolf MJ, Beunen G, Casaer P, Wolf B: Extreme hyperbilirubinemia in Zimbabwean neonates: Neurodevelopmental outcome at 4 months. Eur J Pediatr 156:803, 1997.

Halamek LP, Stevenson DK: Neonatal jaundice and liver disease, in Neonatal Perinatal Medicine: Diseases of the Fetus and Infant, 6th ed, edited by AA Fanaroff, RJ Martin, p 1345. Mosby Year Book, St Louis, 1997.
Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn
Williams Hematology



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