Hemolytic Disease of the Newborn

Background

A French midwife was the first to report hemolytic disease of the newborn (HDN) in a set of twins in 1609. In 1932, Diamond and colleagues described the relationship among fetal hydrops, jaundice, anemia, and erythroblasts in the circulation, a condition later called erythroblastosis fetalis. Levine later determined the cause after Landsteiner and Weiner discovered the Rh blood group system in 1940. In 1953, Chown subsequently confirmed the pathogenesis of Rh alloimmunization to be the result of passage of Rh-positive fetal RBCs after transplacental hemorrhage into maternal circulation that lacked this antigen.

In 1966, 2 groups from the United Kingdom and the United States demonstrated, in a combined study, that anti-D immunoglobulin G (IgG) prophylaxis soon after delivery prevented sensitization in Rh-negative women. The World Health Organization (WHO) technical report in 1971 recommended that a dose of 25 mcg (125 IU) of anti-D immunoglobulin G (IgG) should be given intramuscularly for every 1 mL of fetomaternal hemorrhage of Rh-positive packed RBCs or 2 mL of whole blood.^[1]

In 1998, this recommendation was reinforced by the American Association of Blood Banks and the American College of Obstetrics and Gynecologists with inclusion of prophylaxis at 28 weeks' gestation.^[2] Routine use of Rh IgG prophylaxis resulted in a significant decline in the incidence of RhD alloimmunization,^[3] and erythroblastosis fetalis has become rare. The perinatal effects of maternal Rh alloimmunization are now referred to as hemolytic disease of the fetus and newborn, and fetal manifestations of the disease are more appreciated with newer technologies such as cordocentesis and fetal ultrasonography.

• References

Pathophysiology

Genetics

Although the Rh antibody was and still is the most common cause of severe hemolytic disease of the newborn (HDN), other alloimmune antibodies belonging to Kell (K and k), Duffy (Fya), Kidd (Jka and Jkb), and MNSs (M, N, S, and s) systems do cause severe HDN.^[4] The Rh blood group system uses Fisher-Race nomenclature, and the Rh gene complex consists of 3 genetic loci each with 2 major alleles. They code for 5 major antigens denoted by letters, C, c, E, e, and D. Rh blood group antigens are inherited as determined by at least 2 homologous but distinct membrane-associated proteins. Two separate genes (RhCE and RhD), located on the short arm of chromosome 1, encode Rh proteins. Each gene is 10 exons in length, and a 96% homology between these genes is observed.

Production of 2 distinct proteins from the RHCE gene is due to alternative splicing of messenger RNA. Rh gene complex is described by 3 loci, and, therefore, 8 gene complexes are possible. These complexes are as follows (listed in decreasing order of frequency among whites): CDe, cde, CDE, cDe, Cde, cdE, CDE, and CdE. Expression is limited to red blood cells (RBCs), with an increasing density during their maturation, unlike the ABH system, which exists in a wide variety of tissues. Rh antigen is not expressed on RBC progenitors.

Of individuals who are Rh positive, 45% are homozygous (CDe/CDe), and 55% are heterozygous (CDe/cde) for the RhD gene. The Rh-negative phenotype represents absence of D protein on RBCs and most commonly results from deletion of the RHD gene on both chromosomes. However, the RHD gene has significant heterogeneity, and several inherited mutations and rearrangements in its structure can result in a lack of expressions of the RhD phenotype as well.

Important examples of such mutations include the RHD pseudogene and RHD-CE-D hybrid gene. The former leads to a stop codon in RHD gene and results in a lack of transcription product despite all intact exons. It is found in 70% of South African blacks and in 25% of African Americans. The RHD-CE-D (Ccde) gene is also found in 22% of D-negative African Americans. It also results in an Rh positive genotype but a negative phenotype. Most Caucasians who are RhD negative have a complete deletion of RHD gene whereas only 18% of African blacks and 54% of African Americans who are RhD negative have complete deletion of the gene; the rest have above nonfunctional variants of the RHD gene.^[5]

Beyond the 5 major antigens, more than 100 antigenic variants of Rh group system have been identified. Individuals with these weak-D phenotypes comprise of 2 populations: first group (90%) that expresses normal but reduced quantities of D antigen on the RBC surface, and most cannot be sensitized to produce anti-D. However, the second group (remaining 10%) known as partial-D (eg, Cw, Du) that express partial D epitopes on RBC surface and can make anti-D and rarely experience fatal HDN. The partial D phenotype results from amino acid sunstitution in the active RhD epitope.^[6] Most women with partial-D phenotype are classified as Rh negative on routine testing and are candidates for Rh immune globulin (RhIG). Currently, testing of all Rh-negative women for weak expression of D is not recommended. However, Rh-negative infants born to Rh-negative women should undergo testing to detect the partial-D phenotype so that RhIG can be administeredin theevent ofweak expression.

Frequency of Rh negativity is higher in whites (15%) than in blacks (5%) and Hispanics (8%) and is rare in Eskimos, Native Americans, Japanese, and Asians, especially in Chinese individuals. The paternal heterozygosity determines the likelihood of an Rh-positive child being born to an Rh-negative mother.^[3]

Pathophysiology

The exposure of the Rh-negative mother to Rh-positive red cells occurs as a result of asymptomatic fetomaternal hemorrhage during pregnancy. The Kleihauer-Betke acid elution technique that determines the proportion of fetal RBCs in maternal circulation has shown the incidence of fetomaternal hemorrhage to be 75% of all pregnancies. Incidence and degree of such hemorrhage appears to increase with gestation. Fetomaternal hemorrhage has been documented in 7%, 16%, and 29% of mothers during their first, second and third trimesters, respectively. Risk is also increased in pregnancies complicated by placental abruption, spontaneous or therapeutic abortion, and toxemia, as well as after cesarean delivery and ectopic pregnancy.

Procedures such as amniocentesis, chorionic villus sampling, and cordocentesis also increase the risk of alloimmunization. Because the transplacental hemorrhage is less than 0.1 mL in most pregnancies, most women are sensitized as a result of small, undetectable fetomaternal hemorrhage.

After the initial exposure to a foreign antigen, B-lymphocyte clones that recognize the RBC antigen are established. The maternal immune system initially produces antibodies of the immunoglobulin M (IgM) isotype that do not cross the placenta and later produces antibodies of the IgG isotype that traverse the placental barrier. Predominant antibody subclass appears to be IgG1 in one third of individuals whereas a combination of IgG1 and IgG3 subclasses are found in the remaining individuals.

IgG3 is more efficient in binding to reticuloendothelial cells and causing hemolysis because of its longer hinge region. This is termed the primary response and is dose dependent (documented in 15% of pregnancies with 1 mL of Rh-positive cells in an Rh-negative individual compared with 70% of pregnancies after 250 mL). A repeat exposure to the same antigen rapidly induces the production of IgG. This secondary immune response can be induced with as little as 0.03 mL of Rh-positive RBCs.

Findings from murine models appear to support a potential role for epitope masking, immune deviation and/or antigen modulation in the mechanism of action of IgG-mediated inhibition of erythrocyte alloimmunization.^[7]  Moreover, blends of monoclonal antibodies targeting nonoverlapping epitopes on the RBC surface may have the potential to improve the efficacy of monoclonal antibodies approaching that of polyclonal IgG.

The risk of Rh immunization after the delivery of the first child to a nulliparous Rh-negative mother is 16% if the Rh-positive fetus is ABO compatible with its mother, 2% if the fetus is ABO incompatible, and 2-5% after an abortion. The ABO-incompatible RBCs are rapidly destroyed in the maternal circulation, reducing the likelihood of exposure to the immune system. The degree of Rh sensitization of the mother is directly related to the amount of fetomaternal hemorrhage (ie, 3% with < 0.1 mL compared with 22% with >0.1 mL).

After sensitization, maternal anti-D antibodies cross the placenta into fetal circulation and attach to Rh antigen on fetal RBCs, which form rosettes on macrophages in the reticuloendothelial system, especially in the spleen. These antibody-coated RBCs are lysed by lysosomal enzymes released by macrophages and natural killer lymphocytes and are independent of the activation of the complement system.

Reticulocytosis is noted when fetal Hb deficit exceeds 2 gm/dl compared with gestational age norms. Tissue hypoxia develops as fetal anemia becomes severe. When the hemoglobin (Hb) level drops below 8 g/dL, a rise in umbilical arterial lactate occurs. When the Hb level drops below 4g/dL, increased venous lactate is noted. Hydrops fetalis occurs when fetal Hb deficit exceeds 7 g/dL and starts as fetal ascites and evolves into pleural effusions and generalized edema. The various mechanisms responsible for hydrops are hypoalbuminemia secondary to depressed liver function, increased capillary permeability, iron overload secondary to hemolysis, and increased venous pressures due to poor cardiac function.^[8]

Prolonged hemolysis leads to severe anemia, which stimulates fetal erythropoiesis in the liver, spleen, bone marrow, and extramedullary sites, such as the skin and placenta. In severe cases, this can lead to displacement and destruction of hepatic parenchyma by erythroid cells, resulting in dysfunction and hypoproteinemia. Destruction of RBCs releases heme that is converted to unconjugated bilirubin. Hyperbilirubinemia becomes apparent only in the delivered newborn because the placenta effectively metabolizes bilirubin. HDN due to Kell sensitization results in hemolysis and suppression of erythropoiesis because the Kell antigen is expressed on the surface of erythroid progenitors. This leads to severe fetal disease at a lower maternal antibody titer than in Rhesus disease.

Hemolysis associated with ABO incompatibility exclusively occurs in type-O mothers with fetuses who have type A or type B blood, although it has rarely been documented in type-A mothers with type-B infants with a high titer of anti-B IgG. In mothers with type A or type B, naturally occurring antibodies are of the IgM class and do not cross the placenta, whereas 1% of type-O mothers have a high titer of the antibodies of IgG class against both A and B. They cross the placenta and cause hemolysis in fetus.

Hemolysis due to anti-A is more common than hemolysis due to anti-B, and affected neonates usually have positive direct Coombs test results. However, hemolysis due to anti-B IgG can be severe and can lead to exchange transfusion. Because A and B antigens are widely expressed in various tissues besides RBCs, only a small portion of antibodies crossing the placenta are available to bind to fetal RBCs. Recent analysis of IgG subclass in ABO incompatible direct coombs positive neonates showed IgG2 was predominent antibody which is poorly transferred across placenta and less efficient in causing hemolysis while IgG1 was noted in 22% of neonates and as a group had similar rate of hemolysis and severity of hyperbilirubinemia.^[9]

In addition, fetal RBCs appear to have less surface expression of A or B antigen, resulting in few reactive sites; hence the low incidence of significant hemolysis in affected neonates. This results in hyperbilirubinemia as a predominant manifestation of incompatibility (rather than anemia), and peripheral blood film frequently reveals a large number of spherocytes and few erythroblasts, unlike what is seen in Rh incompatibility (erythroblastosis fetalis), in which blood film reveals a large number of nucleated RBCs and few spherocytes.^[10]

• References

Etiology

In the absence of a positive direct Coombs test result, other causes of pathologic jaundice should be considered,^[11]  including intrauterine congenital infections; erythrocyte membrane defects (eg, hereditary spherocytosis, hereditary elliptocytosis, hereditary pyropoikilocytosis); RBC enzyme deficiencies (eg, glucose-6-phosphate dehydrogenase [G6PD] deficiency, pyruvate kinase deficiency, triosephosphate isomerase deficiency); and nonhemolytic causes (eg, enclosed hemorrhages, hypothyroidism, GI obstruction, and metabolic diseases).

Similarly, hydrops can occur from nonimmune hematologic disorders that cause anemia, such as hemoglobinopathies (eg, α-thalassemia major), cardiac failure due to dysrhythmia, congenital heart defects, and infections (eg, syphilis, cytomegalovirus [CMV], parvovirus^[12] ).

• Common causes of hemolytic disease of the newborn

  • Rh system antibodies
  • ABO system antibodies

   

  

   
• Uncommon causes: Kell system antibodies
• Rare causes

  • Duffy system antibodies
  • MNS and s system antibodies

   

  

   
• No occurrence in hemolytic disease of the newborn

  • Lewis system antibodies
  • P system antibodies

   

  

   

• References

Epidemiology

United States data

Hemolytic disease of the fetus and newborn (HDFN) affects an estimated 3 in 100,000 to 80 in 100,000 patients annually.^[13]

Before the establishment of modern therapy, 1% of all pregnant women developed Rh alloimmunization. Since the advent of routine prophylaxis of at-risk women, incidence of Rh sensitization has declined from 45 cases per 10,000 births to 10.2 cases per 10,000 total births, with less than 10% requiring intrauterine transfusion.^[14]  Alloimmunization due to Kell antigen accounts for 10% of severely affected fetuses.

Currently, anti-D is still one of the most common antibodies found in pregnant women, followed by anti-K, anti-c, and anti-E. Of those fetuses who require intrauterine transfusions, 85%, 10%, and 3.5% were due to anti-D, anti-K, and anti-c, respectively.^[15] ABO incompatibility frequently occurs during the first pregnancy and is present in approximately 12% of pregnancies, with evidence of fetal sensitization in 3% of live births. Less than 1% of births are associated with significant hemolysis.

Race- and sex-related demographics

Incompatibility involving Rh antigens (anti-D or anti-c) occurs in about 10% of all pregnancies among whites and blacks; in contrast, it is very rare in Asian women.

Fetal sex plays a significant role in the degree of response to maternal antibodies. An apparent 13-fold increase is observed in fetal hydrops in RhD-positive male fetuses compared with female fetuses in similarly sensitized pregnancies.^[8]

• References

Prognosis

Overall survival is 85-90% but reduced for hydropic fetuses by 15%. Most survivors of alloimmunized gestation are intact neurologically. Fetal hydrops does not seem to affect long-term outcome.^[16] However, neurologic abnormality has been reported to be closely associated with severity of anemia and perinatal asphyxia. Sensorineural hearing loss may be slightly increased.

Morbidity/mortality

Almost 50 different red cell surface antigens have been found to be responsible for hemolytic disease of fetus and newborn. Only 3 antibodies are associated with severe fetal disease: anti-RhD, anti-Rhc, and anti-Kell(K1). Nearly 50% of the affected newborns do not require treatment, have mild anemia and hyperbilirubinemia at birth, and survive and develop normally. Approximately 25% are born near term but become extremely jaundiced without treatment and either die (90%) or become severely affected by kernicterus (10%). The remaining 25% of affected newborns are severely affected in utero and become hydropic; about half of newborns are affected before 34 weeks' gestation, and the other half are affected between 34 weeks' gestation and term.^[1]

Before any interventions were available, the perinatal mortality rate was 50%. Wallerstein introduced exchange transfusion in 1945 and reduced the perinatal mortality rate to 25%. Later, Chown suggested the early delivery of those severely affected nonhydropic fetuses by 34 weeks' gestation followed by prompt exchange transfusion helped improve survival. The introduction of intraperitoneal transfusion by William Liley in 1963 and intravascular transfusion (IVT) by Rodeck in 1981 reduced the perinatal morbidity and the mortality rate was further reduced to the current rate of 16%.

Mortality rises to 30% with any degree of fetal hydrops. Most fetuses who are able to reverse fetal hydrops after IVT survive, compared with 25% of those in whom fetal hydrops was severe and persisted despite treatment. The overall rate of neurodevelopmental impairment is 10%, which is comparable to that found in the general population, but hearing loss is increased 5-10 fold over the general population in those infants who require in utero therapy for hemolytic disease of the newborn. The LOTUS study in a Dutch population reported neurodevelopmental outcomes in 281 children with hemolytic disease of the fetus treated with IVT at 8 years, showing normal outcome in 94%, cerebral palsy in 2.1%, severe developmental delay in 3.1%, and bilateral deafness in 1%.^[17]  Severe hydrops fetalis was the only independent risk factor identified for poor outcome.

No relationship was noted between global developmental scores and the severity of hemolytic disease of the newborn (as evidenced by such factors as the number of intrauterine transfusions [IUTs], the lowest hematocrit [Hct] level, or the presence of hydrops). Normal neurological outcome is noted in more than 90% of infants even if fetal hydrops noted at the time of the first IUT.^[18]

Exchange transfusion

A study by Smits-Wintjens et al indicated that exchange transfusion in neonates increases the risk of sepsis, severe thrombocytopenia, leukocytopenia, hypernatremia, and hypocalcemia in neonates with hemolytic disease of the newborn (HDN). The study involved 347 newborns with red cell alloimmune hemolytic disease, including 134 who underwent exchange transfusion and 213 who did not. The incidence of complications in the two groups was, respectively, as follows^[19] :

• Proven sepsis: 8% versus 1%
• Severe thrombocytopenia: 63% versus 8%
• Leukocytopenia: 88% versus 23%
• Hypernatremia: 8% versus 0%
• Hypocalcemia: 22% versus 1%

• References

History

Two usual patterns of Rh isoimmunization severity are noted. The disease may remain at the same degree of severity or may become progressively worst with each pregnancy. A history of hydropic birth increases the risk of fetal hydrops in the next pregnancy to 90%; the fetal hydrops occurs at about the same time or earlier in gestation in the subsequent pregnancy. Women at risk for alloimmunization should undergo an indirect Coombs test and antibody titers at their first prenatal visit. If results are positive, obtain a paternal blood type and genotype with serologic testing for other Rh antigens (C, c, E, e).

The paternal zygosity for the D allele is determined from race-specific gene frequency tables that take into account the serology results of Rh antigen expression, ethnicity, and number of previous Rh-positive children.^[20] In the event of unclear ethnicity, quantitative polymerase chain reaction (PCR) of the RHD gene has been used to detect the heterozygous state.^[21] Two such assays, one based on direct amplification of deletion and the other using RHD gene copy number with a reference gene, are available. Recent research revealed quantitative PCR to be highly accurate for detecting a paternal heterozygous state and now is preferred over serologic testing owing to the high frequency of interracial marriages.^[22]

Obtaining serial maternal titers is suggested if the father is homozygous. If the father is heterozygous, determine fetal Rh genotype using PCR for the RHD gene on fetal cells obtained at amniocentesis^[23] or on cell-free DNA in maternal circulation.^[24] The sensitivity and specificity of PCR typing on amniotic fluid is 98.7% and 100%, respectively. However, obtaining maternal blood to rule out a maternal RHD pseudogene (in a Rh-positive fetus) and obtaining paternal blood to rule out RHD gene locus rearrangement (in a Rh-negative fetus) is important to improve the accuracy.^[25] Determining fetal Rh genotype is also possible by performing cordocentesis, which is also called fetal blood sampling (FBS). FBS is associated with a more than 4-fold increase in perinatal loss compared with amniocentesis.

Indicators for severe hemolytic disease of the newborn (HDN) include mothers who have had previous children with hemolytic disease, rising maternal antibody titers, rising amniotic fluid bilirubin concentration, and ultrasonographic evidence of fetal hydrops (eg, ascites, edema, pleural and pericardial effusions, worsening biophysical profile, decreasing hemoglobin [Hb] levels). The major advance in predicting the severity of hemolytic disease was the delta-OD 450 reported by Liley in 1961.^[26] The serial values of deviation from baseline at 450 nm, the wavelength at which bilirubin absorbs light, are plotted on a Liley curve (see the image below) against the gestational weeks. The values above 65% on zone 2 indicate direct fetal monitoring by cordocentesis. Hematocrit (Hct) levels below 30% or a single value in zone 3 are indications for intrauterine transfusion.

View Image

Liley curve. This graph illustrates an example of amniotic fluid spectrophotometric reading of 0.206, which when plotted at 35 weeks' gestation falls ....

The modification of Liley chart was developed by extrapolating the Liley curve^[27] and is used to correct for gestations of less than 27 weeks because bilirubin levels normally peak at 23-25 weeks' gestation in unaffected fetuses (see the image below).^[28]

View Image

Modified Liley curve for gestation of less than 24 weeks showing that bilirubin levels in amniotic fluid peak at 23-24 weeks' gestation.

Another curve was developed by Queenan for management of pregnancies before 27 weeks' gestation (see the image below).^[29]

View Image

Queenan Curve: Modified Liley curve that shows delta-OD 450 values at 14-40 weeks' gestation.

In a recent prospective evaluation, the Queenan curve predicted moderate anemia with a sensitivity of 83% and a specificity of 94%, whereas the sensitivity and specificity for severe anemia were 100% and 79%, respectively.^[30] The delta-OD 450 value that plots out in the intrauterine death risk zone of Queenan curve indicates the need for FBS. A recent comparison of the curves found the Queenan curve to be superior to the Liley curve in overall sensitivity, specificity, and accuracy; when limited to less than 27 weeks' gestation, its sensitivity was higher by 10%, with both having a specificity of 40%.^[31]

• References

Physical Examination

An infant born to an alloimmunized mother shows clinical signs based on the severity of the disease. The typical diagnostic findings are jaundice, pallor, hepatosplenomegaly, and fetal hydrops in severe cases. The jaundice typically manifests at birth or in the first 24 hours after birth with rapidly rising unconjugated bilirubin level. Occasionally, conjugated hyperbilirubinemia is present because of placental or hepatic dysfunction in those infants with severe hemolytic disease. Anemia is most often due to destruction of antibody-coated red blood cells by the reticuloendothelial system, and, in some infants, anemia is due to intravascular destruction. The suppression of erythropoiesis by intravascular transfusion (IVT) of adult Hb to an anemic fetus can also cause anemia. Extramedullary hematopoiesis can lead to hepatosplenomegaly, portal hypertension, and ascites.

Anemia is not the only cause of hydrops. Excessive hepatic extramedullary hematopoiesis causes portal and umbilical venous obstruction and diminished placental perfusion because of edema. Increased placental weight and edema of chorionic villi interfere with placental transport. Fetal hydrops results from fetal hypoxia, anemia, congestive cardiac failure, and hypoproteinemia secondary to hepatic dysfunction. Commonly, hydrops is not observed until the Hb level drops below approximately 4 g/dL (Hct < 15%).^[8]  Clinically significant jaundice occurs in as many as 20% of ABO-incompatible infants.

• References

Laboratory Studies

The diagnosis and management of pregnant women with hemolytic disease of the newborn (HDFN) is based on laboratory and radiographic monitoring.^[13]

Hemolytic disease of the newborn is characterized by one or more of the following clinical presentations^[33] :

• Rapidly progressive severe hyperbilirubinemia or prolonged hyperbilirubinemia
• Positive maternal antenatal antibody findings and/or diagnosis of anemia or fetal hydrops
• Positive neonatal direct Coombs test (direct antiglobulin test)
• Hemolysis on blood film findings

The severity of hematologic abnormalities is directly proportional to the severity of hemolysis and the extent of hematopoiesis. The following abnormalities are observed on complete blood cell (CBC) count findings:

• Anemia: Measurements are more accurate using central venous or arterial samples rather than capillary blood.
• Increased nucleated red blood cells (RBCs), reticulocytosis, polychromasia, anisocytosis, spherocytes, and cell fragmentation

  • The reticulocyte count can be as high as 40% in patients without intrauterine intervention.
  • The nucleated RBC count is elevated and falsely elevates the leukocyte count, reflecting a state of erythropoiesis.
  • Spherocytes (< 40%) are more commonly observed in cases of ABO incompatibility. Glucose does not correct the autohemolysis in ABO incompatibility unlike hereditary spherocytosis.
  • In severe hemolytic disease, schistocytes and burr cells may be observed, reflecting ongoing disseminated intravascular coagulation (DIC).
  • In neonates with sepsis, risk factors for DIC include asphyxia, bleeding, and gram-negative bacterial infection.^[34]
  • A low reticulocyte count is observed in fetuses provided with intravascular transfusion in utero and with Kell alloimmunization.
  • Abnormally elevated mean cell hemoglobin concentration (MCHC) and red cell distribution width (RDW) values should prompt a diagnosis of hereditary spherocytosis.^[35]

   

  

   
• Neutropenia: This condition seems to be secondary to stimulation of erythropoiesis in favor of myelopoiesis. However, neutrophilia can be observed after intrauterine transfusion because of an increase in circulating cytokines (granulocyte-macrophage colony-stimulating factor).
• Thrombocytopenia: This condition is common, especially after intrauterine or exchange transfusions because of platelet-poor blood product and suppression of platelet production in favor of erythropoiesis.

Hypoglycemia is common and is due to islet cell hyperplasia and hyperinsulinism.^[36]  The abnormality is thought to be secondary to release of metabolic byproducts such as glutathione from lysed RBCs. Hypokalemia, hyperkalemia, and hypocalcemia are commonly observed during and after exchange transfusion.

Serologic test findings include the following:

• Indirect Coombs test and direct antibody test results are positive in the mother and affected newborn. Unlike Rh alloimmunization, direct antibody test results are positive in only 20-40% of infants with ABO incompatibility.^[37] In a recent study,^[38] positive direct antibody test findings have a positive predictive value of only 23% and a sensitivity of only 86% in predicting significant hemolysis and need for phototherapy, unless the findings are strongly positive (4+). This is because fetal RBCs have less surface expression of type-specific antigen compared with adult cells. A prospective study has shown that the titers of maternal immunoglobulin G (IgG) anti-A or anti-B may be more helpful in predicting severe hemolysis and hyperbilirubinemia. The sensitivity and specificity of IgG titers of 512 or higher in predicting need for invasive intervention was 90% and 73%, respectively.^[39]  In a retrospective study (2005-2014) in a regional Belgian population, routine testing of maternal serum for relevant erythrocyte antibodies showed that in mother with positive antibodies, significant hyperbilirubinemia was noted more often if cord direct antiglobulin test (DAT) was positive (15% vs 2.6%).^[40]  Significant hyperbilirubinemia was was noted in 7%, 11%, and 27%, respectively, in those with clinically relevant erythrocyte antibodies, anti-A and anti-B.  
• Although the indirect Coombs test result (neonate's serum with adult A or B RBCs) is more commonly positive in neonates with ABO incompatibility, it also has poor predictive value for hemolysis. This is because of the differences in binding of IgG subtypes to the Fc receptor of phagocytic cells and, in turn, in their ability to cause hemolysis.
• IgG2 is more commonly found in maternal serum but has weak lytic activity, which leads to the observation of little or no hemolysis with a positive direct antibody test result. On the other hand, significant hemolysis is associated with a negative direct antibody test result when IgG1 and IgG3 are predominant antibodies, which are in low concentration but have strong lytic activity, crossing to neonatal circulation.
• In newborns with hemolytic disease due to anti-c or anti-C antibodies, direct antibody test results may be negative, and the diagnosis is established after indirect Coombs testing.
• More recent studies on antibody characteristics have shown that lower core fucosylation of Rh-D antibodies while glycosylation and sialylation of anti-c antibodies significantly correlated with disease severity and fetal/neonatal disease.^[41]

Obtain paternal molecular RhD zygosity testing. In addition, fetal RhD genotyping via cell free fetal DNA testing has become increasingly accurate in detecting fetal RhD allosensitization.^[3]

Table. Comparison of Rh and ABO Incompatibility

View Table

See Table

Carboxyhemoglobin (COHb) values measured with a CO-oximeter appear to have the potential to confirm hemolysis in infants with ABO alloimmunization.^[42] In a prospective study of 86 term jaundiced newborn infants, with or without hemolysis, and healthy controls, infants with ABO hemolytic disease had higher COHb values compared to the healthy control infants and newborns with hyperbilirubinemia without hemolytic disease, but no significantly higher value in COHb results was found between the hyperbilirubinemia without hemolytic disease group and the healthy control group. A cut-off value of 1.7% COHb was 72% sensitive and 97% specific for confirming hemolysis in ABO alloimmunization.^[42]

• References

Imaging Studies

High-resolution ultrasonography has been a major advance in detection of early hydrops and has also reduced the fetal trauma and morbidity rate to less than 2% during percutaneous umbilical blood sampling (PUBS) and placental trauma during amniocentesis. High-resolution ultrasonography has been extremely helpful in directing the needle with intraperitoneal transfusion (IPT) and intravascular transfusion (IVT) in fetal location.

• References

Approach Considerations

The following may be indicated in patients with hemolytic disease of newborn (HDN):

• The stabilization of a hydropic newborn requires a high level of intensive coordinated management by a neonatal team well prepared for the possibly affected infant.
• In general, immediate intubation followed by draining of pleural effusions and ascites results in immediate improvement in respiratory gas exchange.
• A cautious correction of anemia with packed red blood cells (RBCs) or by exchange transfusion is necessary to prevent circulatory overload.
• These neonates have normal blood volume but elevated central venous pressure.
• A close monitoring of metabolic status (eg, watching for hypoglycemia, hypocalcemia, hyperkalemia, acidosis, hyponatremia, renal failure) is absolutely essential to achieve a successful outcome.
• Despite of the first use of phototherapy by Cremer and associates more than 40 years ago, no standard method for delivering phototherapy is yet available.

  • Phototherapy units differ widely with respect to the type and size of lamps used. The efficacy of phototherapy depends on the spectrum of light delivered, the blue-green region (425-490 nm) of visible light being the most effective; irradiance (µW/cm²/nm); and surface area of the infant exposed.
  • High-intensity phototherapy first described by Tan in 1977 uses irradiance greater than 25 µW/cm²/nm up to 40 µW/cm²/nm when a dose-response relationship to bilirubin degradation reaches a plateau.
  • Nonpolar bilirubin is converted into 2 types of water-soluble photoisomers as a result of phototherapy. The initial and most rapidly formed configurational isomer 4z, 15e bilirubin accounts for 20% of total serum bilirubin level in newborns undergoing phototherapy and is produced maximally at conventional levels of irradiance (6-9 µW/cm²/nm).
  • The structural isomer lumirubin is slowly formed, and its formation is irreversible and is directly proportional to the irradiance and surface area of skin exposed to phototherapy. Lumirubin is the predominant isomer formed during high-intensity phototherapy.^[43]  Decrease in bilirubin is mainly the result of excretion of these photoproducts in bile and removal via stool. In the absence of conjugation, these photoisomers can be reabsorbed by way of the enterohepatic circulation and diminish the effectiveness of phototherapy.
  • Phototherapy implementation guidelines were addressed in clinical practice guidelines published by the American Academy of Pediatrics.^[44]  The recommendations are as follows:
    • The guidelines are based on total serum bilirubin levels and the direct fraction should not be subtracted from the total unless it is more than 50% of the total serum bilirubin level.
    • Intensive phototherapy should be started for babies with hemolytic disease. This implies the use of irradiance in the 430-490 nm band of more than 30 µW/cm²/nm delivered to as much of the infant's surface area as possible. This can be accomplished using special blue fluorescent tubes that are labeled F20T12/BB or TL52/20W and positioning them 10-15 cm above the infant. When fluorescent tubes are used, they should be brought as close to the infant as possible to increase irradiance. However, when halogen spotlights are used, the distance above the infant should be as per the manufacturer's instructions because spotlights can cause burns. Phototherapy lights emit minimal ultraviolet (UV) radiation that does not cause erythema and is completely absorbed by the acrylic Plexiglas covering of the tubes.
    • Irradiance should be measured using radiometers recommended by the manufacturers of phototherapy systems at multiple sites on the infant's body surface illuminated by the phototherapy lamp and the measurements averaged.
    • The infant should be in the bassinet, and the sides should be lined with white cloth or aluminum foil to expose more surface area. The exposed surface area is increased by the use of 1-2 fiberoptic pads that should be placed under the infant or by the use of BiliBed or Bili-Bassinet, which provides phototherapy from above and below. The diaper should be removed if bilirubin is approaching exchange levels.
    • The serum bilirubin declines by 0.5-1 mg/dL in the first 4-8 hours on intensive phototherapy and should be measured in 2-3 hours to document the effectiveness.
    • If the serum bilirubin level continues to rise despite intensive phototherapy or is within 2-3 mg/dL of exchange level, administer intravenous immunoglobulin (IVIG) at 0.5-1 g/kg over 2 hours and repeat every 12 hours if needed.
    • High-dose IVIG 1 g/kg given early in high-risk neonates with rapid rise of bilirubin level (>0.5 mg/kg/h) and worsening anemia (hemoglobin [Hb] < 2 g/dL) despite intensive phototherapy, is be able to eliminate the need for exchange transfusion and to reduce duration of phototherapy. The number needed to treat (NNT) is 6.^[44]
  • Phototherapy is indicated in the term infant with hemolytic disease of the newborn immediately after birth due to Rh disease and due to ABO incompatibility as follows:^[45]
    • Unborn (cord blood): Total serum bilirubin level of more than 3.5 mg/dL
    • Age less than 12 hours: Total serum bilirubin level of more than 10 mg/dL
    • Age less than 18 hours: Total serum bilirubin level of more than 12 mg/dL
    • Age less than 24 hours: Total serum bilirubin level of more than 14 mg/dL
    • Age 2-3 days: Total serum bilirubin level of more than 15 mg/dL
    • Immediately after birth in all preterms who weigh less than 2500 g

   

  

   
• Exchange transfusion removes circulating bilirubin and antibody-coated RBCs, replacing them with RBCs compatible with maternal serum and providing albumin with new bilirubin binding sites. The process is time consuming and labor intensive but remains the ultimate treatment to prevent kernicterus. The process involves the placement of a catheter via the umbilical vein into the inferior vena cava and removal and replacement of 5- to 10-mL aliquots of blood sequentially, until about twice the volume of the neonate's circulating blood volume is reached (ie, double-volume exchange).
• This process removes approximately 70-90% of fetal RBCs. The amount of bilirubin removed directly varies with the pretransfusion bilirubin level and amount of blood exchanged. Because most of the bilirubin is in the extravascular space, only about 25% of the total bilirubin is removed by an exchange transfusion. A rapid rebound of serum bilirubin level is common after equilibration and frequently requires additional exchange transfusions. However, continued hemolysis and anemia in spite of multiple exchange transfusions and negative direct antiglobulin test (DAT), should raise the possibility of absorption of IgG anti-D acquired from maternal breast milk leading to hyporegenerative anemia caused by ongoing hemolysis of erythroid precursor and marrow supression.^[46]
• The indications for exchange transfusion are controversial, except for the fact that severe anemia and the presence of a rapidly worsening jaundice despite optimal phototherapy in the first 12 hours of life indicate the need for exchange transfusion. In addition, the presence of conditions that increase the risk of bilirubin encephalopathy lowers the threshold of safe bilirubin levels.
• Guidelines for exchange transfusion in neonates with hemolytic disease of the newborn are as follows:^[47]

  • Total serum bilirubin level of more than 20 mg/dL: Weight more than 2500 g (healthy)
  • Total serum bilirubin level of more than 18 mg/dL: Weight more than 2500 g (septic)
  • Total serum bilirubin level of more than 17 mg/dL: Weight 2000-2499 g
  • Total serum bilirubin level of more than 15 mg/dL: Weight 1500-1999 g
  • Total serum bilirubin level of more than 13 mg/dL: Weight 1250-1499
  • Total serum bilirubin level of 9-12 mg/dL: Weight less than 1250

   

  

   
• The following are indications for exchange transfusion:^[48]

  • Severe anemia (Hb < 10 g/dL)
  • Cord bilirubin above 4 mg/dL.
  • Rate of bilirubin rises more than 0.5 mg/dL despite intensive phototherapy
  • Severe hyperbilirubinemia^[44]
  • Serum bilirubin-to-albumin ratio exceeding levels that are considered safe

   

  

   
• Exchange transfusion should be considered in newborns born at more than 38 weeks' gestation with a bilirubin-to-albumin ratio of 7.2 and in newborns born at 35-37 weeks' gestation with a bilirubin-to-albumin ratio of 6.8. Exchange transfusion is not free of risk, with the estimated morbidity rate at 5% and the mortality rate as high as 0.5%. Apnea, bradycardia, cyanosis, vasospasm, and hypothermia with metabolic abnormalities (eg, hypoglycemia, hypocalcemia) are the most common adverse effects.
• IVIG has been shown to reduce the need for exchange transfusion in hemolytic disease of the newborn due to Rh or ABO incompatibility. The number needed to treat to prevent one exchange transfusion was noted to be 2.7 and was estimated to be 10, if all the infants with strongly positive direct Coombs test were to receive the medication.^{[49, 50]}  In addition, it also reduced the duration of hospital stay and phototherapy.^[51]  Although it was very effective as a single dose, multiple doses were more effective in stopping the ongoing hemolysis and reducing the incidence of late anemia.
• A randomized, controlled trial by Smits-Wintjens et al, however, failed to show the benefit of prophylactic single-dose IVIG at 0.75 g/kg within 4 hours of life in severely sensitized neonates with prior IUT due to Rh alloimmunization.^[52]  Although IVIG has been proven to be safe, a retrospective review reported almost 30-times increased risk of necrotizing enterocolitis (NEC) in late preterm and term infants.^[53]
• Tin-mesoporphyrin in a dose of 4.5 mg/kg (6 µmole/kg) was used in an infant with persistent hemolysis due to Rh alloimmunization to prevent need for further phototherapy, without any adverse effects.^[54]

• References

Medical Care

Management of maternal alloimmunization

As a rule, serial maternal antibody titers are monitored until a critical titer of 1:32, which indicates that a high risk of fetal hydrops has been reached. At this point, the fetus requires very intense monitoring for signs of anemia and fetal hydrops. In Kell alloimmunization, hydrops can occur at low maternal titers because of suppressed erythropoiesis, and, thus, a titer of 1:8 has been suggested as critical. Hence, delta-OD 450 values are also unreliable in predicting disease severity in Kell alloimmunization.^[55]

Maternal titers are not useful in predicting the onset of fetal anemia after the first affected gestation. Large differences in titer can be seen in the same patient between different laboratories, and a newer gel technique produces higher titer results than the older tube method. Therefore, standard tube methodology should be used to determine critical titer, and a change of more than 1 dilution represents a true increase in maternal antibody titer. For all the antibodies responsible for hemolytic disease of the newborn (HDN), a 4-fold increase in any antibody titer is typically considered a significant change that requires fetal evaluation.^[56]

When indicated, amniocentesis can be performed as early as 15 weeks' gestation (rarely needed in first affected pregnancy before 24 weeks' gestation) to determine fetal genotype and to assess the severity. Maternal and paternal blood samples should be sent to the reference laboratory with amniotic fluid sample to eliminate false-positive results (from maternal pseudogene or Ccde gene) and false-negative results (from a rearrangement at the RHD gene locus in the father).

Fetal Rh-genotype determination in maternal plasma has become routine in many European countries and is being offered in the United States.^[57] Fetal cell-free DNA accounts for 3% of total circulating maternal plasma DNA, is found as early as 38 days of gestation, and is derived from apoptosis of the placental cytotrophoblast layer. The mean half-life of circulating fetal DNA is on average less than 30 minutes, and maternal plasma is subjected to filtration and microcentrifugation to remove all cellular elements before testing. This eliminates false-positive results from engrafted fetal cells of previous pregnancies in maternal lymphoid organs.

Cell-free fetal DNA is subjected to real-time polymerase chain reaction (PCR) for the presence of RHD gene–specific sequences and has been found to be accurate in 99.5% of cases. The SRY gene (in the male fetus) and DNA polymorphisms in the general population (in the female fetus) are used as internal controls to confirm the fetal origin of the cell-free DNA.^[20] A panel of 92 single-nucleotide polymorphisms (SNPs) is compared between maternal sample from buffy coat and plasma. A difference of more than 6 SNPs confirms presence of fetal DNA and the validity of the test in a female fetus.^[57]  False-negative results being most undesirable and consequential are due to partial or weak D phenotypes. They are detected by using at least two RHD-specific exon primers and run in duplicates.^[58]

Fortunately, cell-free fetal DNA testing for determining the genotype for other red blood cell antigens such as c,C, e, E and Kell is also now found to be highly reliable and accurate.^[58]

Serial amniocentesis is begun at 10-14 day intervals to monitor the severity of the disease in the fetus. All attempts should be made to avoid transplacental passage of needle which can lead to fetomaternal hemorrhage (FMH) and a further rise in antibody titer. Serial delta-OD 450 values are plotted on the Queenan chart or the extended Liley chart to evaluate the risk of fetal hydrops. Early ultrasonography is performed to establish correct gestational age. Frequent ultrasonographic monitoring is also performed to assess fetal well-being and to detect moderate anemia and early signs of hydrops.

The peak systolic middle cerebral artery (MCA) Doppler velocity has proved to be a reliable screening tool to detect fetal anemia and has replaced amniocentesis. The MCA is easily visualized with color-flow Doppler; pulsed Doppler is then used to measure the peak systolic velocity just distal to its bifurcation from the internal carotid artery. Because the MCA velocity increases with advancing gestational age, the result is reported in multiples of median (MOMs). In recent studies, the sensitivity for detection of moderate and severe fetal anemia has been proven to be 100%, with a false-positive rate of 10% at 1.5 MOM.^[59] It has been shown to reduce the need for invasive diagnostic procedures such as amniocentesis and cordocentesis by more than 70%.^[59]

MCA Doppler studies can be started as early as 18 weeks' gestation but are not reliable after 35 weeks' gestation.^[60]  It has also been used to time the subsequent fetal transfusion and to diagnose anemia from multiple causes, such as in twin-twin transfusion. The MCA slope from 3-weekly readings is now used to predict fetal risk for severe anemia (see the image below).^[61]

View Image

Slopes for peak systolic velocity in middle cerebral artery (MCA) for normal fetuses (dotted line), mildly anemic fetuses (thin line), and severely an....

With acquisition of experience in performing MCA Doppler study, serial amniocentesis for detecting fetal anemia has been used to lesser extent.^[62]

During the period when intrauterine peritoneal transfusion was the only means of treatment, newborns were routinely delivered at 32 weeks' gestation. This approach resulted in a high incidence of hyaline membrane disease and exchange transfusions. With the advent of intravascular transfusion (IVT) in utero, the general approach to the severely affected fetus is to perform IVT as required until 35 weeks' gestation, with delivery planned at term. Establishment of lung maturity is difficult in these fetuses because of contamination of amniotic fluid with residual blood during transfusion; however, if delivery is planned prior to 34 weeks' gestation, maternal steroid administration to enhance fetal lung maturity is indicated.

In addition, excess amniotic fluid bilirubin levels cause false elevation on the fluorescence depolarization TDx fetal lung maturity test, version II (TDX-FLMII); therefore, other tests to determine fetal lung maturity should be used, such as infrared spectroscopy, lamellar body count, phosphatidylglycerol quantitation or lecithin/sphingomyelin (L/S) ratio.

Liley first described intraperitoneal transfusion (IPT) in 1963. A Tuohy needle is introduced into the fetal peritoneal cavity under ultrasonographic guidance. An epidural catheter is threaded through the needle. A radiopaque medium is injected into the fetal peritoneum. The proper placement is confirmed by delineation outside of bowel or under the diaphragm or by diffusion in fetal ascites. Packed red blood cells (RBCs) at a hemotcrit (Hct) of 75-80% that are CMV-negative, less than 4-days-old, group O, Rh-negative, Kell-negative, leukoreduced, irradiated with 25 Gy to prevent graft versus host disease, and cross-matched with maternal serum are injected in 10-mL aliquots to a volume calculated by the following formula:^[1]

IPT volume = (gestation in weeks - 20) × 10 mL

Residual hemoglobin (Hb) in the fetus is estimated to allow for proper spacing of IPT and selection of gestation of delivery by the following formula:

Hb g/dL = 0.85/125 × a/b × 120 - c/120

In the formula, a is the amount of donor RBC Hb transfused, b is the estimated fetal body weight, and c is the interval in days from the time of transfusion to the time of donor Hb estimation.

IPT is repeated when the fetal Hb is estimated to have dropped to 10 g/dL. Usually, a second IPT is performed 10 days after the first transfusion in order to raise the Hb above 10 g/dL. Then another transfusion is performed every 4 weeks until the time of planned delivery at 34-35 weeks' gestation. Fetal diaphragmatic movements are necessary in order for absorption of RBC to occur. This approach is of no value for a moribund nonbreathing fetus. Maternal complications include infection and transplacental hemorrhage, whereas fetal complications are overtransfusion, exsanguination, cardiac tamponade, infection, preterm labor, and graft versus host disease. Survival rates after IPT approached approximately 75% with the help of ultrasonography.

Direct IVT has become a preferred route of fetal intervention because of the higher rate of complications and limited effectiveness of IPT in a hydropic fetus. Rodeck first successfully performed IVT in 1981. With ultrasonographic guidance, a 20-22 gauge needle is introduced into an umbilical vein at the cord insertion into the placenta or into its intrahepatic portion, and a fetal blood sample is obtained. The blood sample is confirmed to be of fetal origin by rapid alkaline denaturation test. All the relevant fetal tests (eg, blood type, direct antibody test, reticulocyte count, platelet count, Hb level, Hct level, serum albumin level, erythropoietin level) are performed. If the Hb level is less than 11 g/dL or if the Hct level is less than 30%, an IVT is started. The position of the needle is confirmed by noting the turbulence in the fetal vessel on injection of saline. The fetus is frequently paralyzed with pancuronium and given fentanyl 10 mcg/kg to prevent the displacement of the needle by fetal movements. Maternal medication varies from local anesthetic only, to routine indomethacin and conscious sedation, to spinal epidural analgesia.^[41]

The transfusion is performed in 10-mL aliquots to a volume of approximately 50 mL/kg estimated body weight using ultrasonography or until an Hct level of 40% is reached. The procedure is promptly discontinued if cardiac decompensation is noted on ultrasonography findings. Severely anemic fetuses do not tolerate acute correction of their Hct to normal values, and the initial Hct should not be increased by more than 4-fold at the time of first IVT. They should then be monitored every 2-7 days. The IVT is repeated when it reaches a value that reflects critical anemia in the fetus. A loss of 1% of transfused cells per day can be anticipated.^[20]

Some centers perform repeat transfusion at intervals of 10 days, 2 weeks, and every 3 weeks. Others transfuse based on an anticipated decline in fetal hemoglobin of 0.4 g/dL/day, 0.3 g/dL/day, and 0.2 g/dL/day for first, second, and third transfusion intervals, respectively.^[63] The peak systolic MCA velocity has been used to time the second transfusion, with a threshold of 1.32 MOM.^[64] After the first intrauterine transfusion, the presence of red blood cells with adult hemoglobin suppress erythropoiesis and improve oxygen delivery, which is responsible for the poor correlation between peak MCA velocity and severity of fetal anemia. Some centers have found a beneficial effect of combined IVT and IPT transfusion on interval to repeat transfusion.^[25]  

In addition to the complications of IPT, transient fetal bradycardia, cord hematoma, umbilical vein compression, and fetal death have been reported during IVT. However, IVT has many advantages, including immediate correction of anemia and resolution of fetal hydrops, reduced rate of hemolysis and subsequent hyperinsulinemia, and acceleration of fetal growth for nonhydropic fetuses who are often growth retarded. IVT is the only intervention available for moribund hydropic fetuses and those with anterior placenta. The risk of fetal loss is about 0.8% with IVT versus 3.5% per procedure for IPT, and the overall survival rate is 88%.

Recently washed maternal RBCs have been successfully used as a source of antigen-negative RBCs in the event of rare incompatibility but also have been routinely used because of benefits such as decreased risk for sensitization to new red cell antigens, a longer circulating half-life being fresh, and decreased risk of transmission of viral agents.^[65] Mother can donate a unit of red cells after the first trimester.

In the event of pulmonary immaturity and delta-OD 450 in the affected zone of the Queenan curve, oral administration of 30 mg of phenobarbital to the mother 3 times per day, followed by induction in one week, reduces the need for exchange transfusion in the affected neonate.^[66] Excellent algorithms for management of the first affected pregnancy and the pregnancy in a mother with previously affected fetus are outlined in a review by Moise (see the images below).^[67]

View Image

Management of first affected pregnancy.

View Image

Management of pregnant women with previously affected fetus.

Initial attempts to suppress Rh antibody production with Rh hapten, Rh-positive RBC stroma, and administration of promethazine were unsuccessful. Extensive plasmapheresis with partial replacement using 5% albumin (therapeutic plasma exchange) and intravenous immunoglobulin (IVIG) or the administration of IVIG at 1 g/kg body weight weekly has been shown to be moderately effective. The mechanism of action appears to be blockage of Fc receptors in the placenta, reducing antibody transport across to the fetus, Fc receptors on the phagocytes in the fetal reticuloendothelial system, and feedback inhibition of maternal antibody synthesis. However, antibody-dependent cell-mediated cytotoxicity and rebound elevation of antibody concentration, alteration of placental blood flow during the procedure, and postpartum hemorrhage have been noted after plasma exchange.^[41]

A more recent retrospective study comprising 5 pregnant women with severe HDFN due to RBC alloimmunization reported successful treatment with a combined regimen of therapeutic plasma exchange, IVIG, and intrauterine transfusion (IUT) early in the pregnancies.^[68]  The women underwent 3 plasma exchange procedures during weeks 10-13 of pregnancy, following by weekly IVIG infusions; the fetuses received RBC units that fully matched the maternal phenotype to the D, C, E, K, Fy, Jk, and S antigen groups. All the women delivered healthy infants at 33-38 weeks' gestation.^[68]

However, these techniques only postpone the need for percutaneous umbilical blood sampling (PUBS) and IVT until 20-22 weeks' gestation, when these procedures can be performed at a more acceptable risk. A review of IVIG use shows its usefulness in preventing the onset of fetal hydrops and in delaying the need for IUT.^[69] Thus, a combined approach of plasmapheresis that starts at 12 weeks' gestation 3 times in that week, followed by IVIG at a loading dose of 2 g/kg after the third plasmapheresis, and then continued IVIG 1 g/kg/wk until 20 weeks' gestation has been suggested for at-risk fetuses prior to 20 weeks' gestation and can also be used later in gestation if IVT cannot be performed or if hydrops is unresponsive to IVT.^[41]

One report indicated that treatment of fetuses with severe alloimmunization using IVT combined with fetal IVIG therapy at 1 g/kg/dose starting from the third IVT helped in reducing the frequency of IVT and improving signs of hydrops.^[70] A case report shows successful treatment of severe anemia and hydrops in a fetus with alloimmunization due to anti-M antibody with fetal intraperitoneal IVIG injections 2 g/kg given weekly starting 30 weeks.^[71] However, this was a case report, and a randomized controlled trial is needed before this can become standard of care.

Similar regimens of tests and treatment are used in the management of pregnancies affected by nonRhD alloimmunization, such as anti-Rhc, anti-K (K1), and anti-M. Once the mother is diagnosed with an antibody associated with hemolytic disease, an indirect Coombs titer is performed, along with paternal testing for involved antigen and zygosity. Maternal titers are repeated (monthly until 28 weeks' gestation and then every 2 wk) until a threshold for fetal anemia is reached (1:8 for Kell and 1:32 for rest).

Fetal antigen typing is performed via amniocentesis or cell-free fetal DNA in maternal plasma if the father is found to be heterozygous (100% for K1, 65% for M). When the fetus is known to be antigen positive, surveillance for severe fetal anemia is performed, with weekly MCA Doppler screening as early as 16-18 weeks and IUT is carried out if it exceeds 1.5 MOM with a delivery by 38 weeks' gestation.^[72]

Maternal alloantibodies to paternal leukocytes have been shown to result in Fc blockade and to reduce the severity of fetal hemolytic anemia. This may be used in the future.

Management of the sensitized neonate

Mild hemolytic disease accounts for 50% of newborns with positive direct antibody test results. Most of these newborns are not anemic (cord hemoglobin [Hb] >14 g/dL) and have minimal hemolysis (cord bilirubin < 4 mg/dL). Apart from early phototherapy, they require no transfusions. However, these newborns are at risk of developing severe late anemia by 3-6 weeks of life. Therefore, monitoring their Hb levels after hospital discharge is important.

Moderate hemolytic disease accounts for approximately 25% of affected neonates. Moderate hemolytic disease of newborn is characterized by moderate anemia and increased cord bilirubin levels. These infants are not clinically jaundiced at birth but rapidly develop unconjugated hyperbilirubinemia in the first 24 hours of life. Peripheral smear shows numerous nucleated RBCs, decreased platelets, and, occasionally, a large number of immature granulocytes. These newborns often have hepatosplenomegaly and are at risk of developing bilirubin encephalopathy without adequate treatment. Early exchange transfusion with type-O Rh-negative fresh RBCs with intensive phototherapy is usually required. Use of IVIG in doses of 0.5-1 g/kg in a single or multiple dose regimen have been able to effectively reduce need for exchange transfusion.^[73]

A prospective randomized controlled study has shown early high-dose IVIG 1 g/kg at 12 hours of age to reduce duration of phototherapy and hospital stay and to prevent exchange transfusion in neonates with moderate-to-severe Rh isoimmunization.^[51] These newborns are also at risk of developing late hyporegenerative anemia of infancy at 4-6 weeks of life. However, one randomized double-blind placebo-controlled trial failed to show the benefit of prophylactic IVIG therapy 0.75 g/kg within 4 hours of age in severely affected neonates who were treated with intrauterine transfusion for Rh isoimmunization.^[52]

Severe hemolytic disease accounts for the remaining 25% of the alloimmunized newborns who are either stillborn or hydropic at birth. The fetal hydrops is predominantly caused by a capillary leak syndrome due to tissue hypoxia, hypoalbuminemia secondary to hepatic dysfunction, and high-output cardiac failure from anemia. About half of these fetuses become hydropic before 34 weeks' gestation and need intensive monitoring and management of alloimmunized gestation as described earlier. Mild hydrops involving ascites reverses with IVTs in only 88% of cases with improved survival but severe hydrops causing scalp edema and severe ascites and pleural effusions reverse in 39% of cases and are associated with poor survival.

Management of ABO incompatibility

Management of hyperbilirubinemia is a major concern in newborns with ABO incompatibility. The criteria for exchange transfusion and phototherapy are similar to those used in Rh alloimmunization. IVIG has also been very effective when administered early in the course. Tin (Sn) porphyrin a potent inhibitor of heme oxygenase, the enzyme that catalyzes the rate-limiting step in the production of bilirubin from heme, has been shown to reduce the production of bilirubin and reduce the need for exchange transfusion and the duration of phototherapy in neonates with ABO incompatibility.

Tin or zinc protoporphyrin or mesoporphyrins have been studied in newborns. They must be administered intramuscularly in a dose based on body weight, and their effectiveness appears to be dose related in all gestations.^[74] Their possible toxic effects include skin photosensitization, iron deficiency, and possible inhibition of carbon monoxide production. Their use in Rh hemolytic disease of newborn has not been reported. Their routine use cannot be recommended yet because of lack of long-term safety data.

• References

Complications

The 2 major complications of hemolytic disease of the newborn are bilirubin encephalopathy (kernicterus) and late anemia of infancy.

Bilirubin encephalopathy

Note the following:

• Before the advent of exchange transfusion, kernicterus affected 15% of infants born with erythroblastosis. Approximately 75% of these neonates died within 1 week of life, and a small remainder died during the first year of life. Survivors had permanent neurologic sequelae and were thought to have accounted for 10% of all patients with cerebral palsy (CP).
• The mechanism by which unconjugated bilirubin enters the brain and damages it is unclear. Bilirubin enters the brain as lipophilic free bilirubin unbound to albumin, as supersaturated bilirubin acid that precipitates on lipid membrane in low pH, or as a bilirubin-albumin complex that transfers bilirubin to tissue by direct contact with cellular surface. The blood-brain barrier is comprised of ATP-dependent transport proteins and pumps free bilirubin from the brain back into plasma and maintains the concentration gradient of unconjugated bilirubin. A damaged blood-brain barrier enhances the entry and fails to remove all forms of bilirubin into the brain, which is especially important in preterm neonates with respiratory acidosis and vascular injury.
• Bilirubin has been postulated to cause neurotoxicity via 4 distinct mechanisms^[43] : (1) interruption of normal neurotransmission (inhibits phosphorylation of enzymes critical in release of neurotransmitters), (2) mitochondrial dysfunction, (3) cellular and intracellular membrane impairment (bilirubin acid affects membrane ion channels and precipitates on phospholipid membranes of mitochondria), and (4) interference with enzyme activity (binds to specific bilirubin receptor sites on enzymes).
• The pathologic findings include characteristic staining and neuronal necrosis in basal ganglia (especially the globus pallidus and subthalamic nucleus), hippocampal cortex (especially the CA2 sector), brainstem nuclei (especially the auditory, vestibular, and oculomotor), and cerebellum (especially Purkinje cells). The cerebral cortex is generally spared. About half of these neonates also have extraneuronal lesions, such as necrosis of renal tubular, intestinal mucosal, and pancreatic cells.
• Clinical signs of bilirubin encephalopathy typically evolve in 3 phases. Phase 1 is marked by poor suck, hypotonia, and depressed sensorium. Fever and hypertonia are observed in phase 2, and, at times, the condition progresses to opisthotonus. Phase 3 is characterized by high-pitched cry, hearing and visual abnormalities, poor feeding, and athetosis.
• Long-term sequelae include choreoathetoid CP, upward gaze palsy, sensorineural hearing loss, dental enamel hypoplasia of the deciduous teeth, and, less often, mental retardation. The abnormal or reduced auditory brainstem response of wave I (auditory nerve) and wave II and V (auditory brainstem nuclei), depicted as decreased amplitudes, and increased interval I-III and I-V characterize phase I of early, but reversible, encephalopathy. Subtle bilirubin encephalopathy that consists of either cognitive dysfunction, isolated hearing loss, or movement disorder has been described in absence of kernicterus and better correlates with free bilirubin levels.
• Currently, the mortality rate stands at 50% in term newborns, but mortality is nearly universal in the preterm population who may simply appear ill without signs specific for kernicterus. Research has indicated that bilirubin production rates may be the critical piece of information identifying jaundiced infants at risk of neurotoxicity. A high bilirubin production rate is thought to result in rapid transfer of bilirubin to tissue, causing high tissue load, in which case any small further increase has great potential to enter the brain. Because the total serum bilirubin represents not only bilirubin production but also distribution and elimination, it is not an absolute indicator of risk of kernicterus. Techniques have been developed to measure the bilirubin production rates accurately and noninvasively using end-tidal carbon monoxide measurement and percutaneous measurement of carboxyhemoglobin.

Late anemia of infancy

Note the following:

• Infants with significant hemolytic disease often develop anemia in the first month of life and frequently (50%) require packed RBC transfusion. The anemia appears to be due to several factors including suppression of fetal erythropoiesis from transfusion of adult Hb during intrauterine or exchange transfusion, resulting in low erythropoietin levels and reticulocyte count.
• Continued destruction of neonatal RBCs by high titers of circulating maternal antibodies also contributes the development of anemia. Weekly Hcts and reticulocyte count need to be monitored after discharge until renewed erythropoiesis is noted. Administration of recombinant human erythropoietin (rh-EPO) has been shown to minimize the need for transfusion in these newborns.^[75]

Potential complications of exchange transfusion include the following (despite declining frequency of exchange transfusion, adverse events and complications have remained stable in most recent reports)^{[76, 77]} :

• Cardiac: Arrhythmia, volume overload, congestive failure, and arrest
• Hematologic: Overheparinization, neutropenia, thrombocytopenia, and graft versus host disease
• Infectious: Bacterial, viral (cytomegalovirus [CMV], human immunodeficiency virus [HIV], hepatitis), and malarial
• Metabolic: Acidosis, hypocalcemia, hypoglycemia, hyperkalemia, and hypernatremia
• Vascular: Embolization, thrombosis, necrotizing enterocolitis, and perforation of umbilical vessel
• Systemic: Hypothermia

• References

Prevention

Consider the following in patients with hemolytic disease of the newborn (HDN):

• Rh immune globin (RhIG) was licensed in 1968 in North America after several studies demonstrated its effectiveness in preventing Rh alloimmunization when administered to the mother within 72 hours of delivery. The current standard is to administer RhIG to all unsensitized Rh-negative women at 28 weeks' gestation with an additional dose administered soon after birth if the infant is Rh-positive, irrespective of the ABO status of the baby. RhIG is not indicated for mothers with weak or partial D status because most are not at risk for alloimmunization.^[60]
• The standard dose of RhIG is 300 mcg and is increased (300 mcg for every 25 mL of fetal blood in maternal circulation) based on the amount of fetomaternal hemorrhage, which can be quantified using the Kleihauer-Betke technique. Because only 50% of pregnancies with excess fetomaternal hemorrhage can be identified by clinical risk factors, routine screen for excess fetomaternal hemorrhage (FMH) is undertaken in all Rh negative women. However, if the incidence of excess FMH is 0.6%, the maximum risk of sensitization is 0.1%, suggesting routine assessment for excess FMH may not be justified.
• Also administer RhIG to unsensitized Rh-negative women after any event known to be associated with transplacental hemorrhage such as spontaneous or elective abortion, ectopic pregnancy, amniocentesis, chorionic villous sampling, fetal blood sampling (FBS), hydatiform mole, fetal death in late gestation, blunt abdominal trauma, and external cephalic version. The indications for first trimester threatened abortion and ectopic pregnancy with no cardiac activity are not cost effective and are left to the clinician.^[21]
• No more than 5 units of RhIG should be given by intramuscular route in 24-hour period. An intravenous preparation is now available for administration of large doses. If RhIG was inadvertently omitted after delivery, the protection can still be offered if given within first 4 weeks. A repeat dose is not needed if delivery occurs within 3 weeks after administration of RhIG during antenatal period. The current incidence of Rh immunization stands at 0.1% with the above recommendations.
• Most RhIG is derived from human plasma obtained from sensitized women or male donors sensitized with RhD positive cells. Because it is a blood product, it has risks of transmission of viral infections such as hepatitis C and may not be acceptable in some religious denomination. Hence 2 monoclonal anti-D antibodies derived from recombinant technology, BRAD-1 and BRAD-3, are being tested in clinical trials. A new novel polyclonal recombinant antibody, rozrolimupab has also been tested in phase I and II clinical trials with no adverse effects.^[78]

• References

Intravenous immunoglobulin (Gamimune, Gammagard, Sandoglobulin, Gammar-P)

• Dosing, Interactions, etc.

Clinical Context:  Several studies have reported success in minimizing the need for exchange transfusion in severe HDN with IVIG. Effective adjunct to phototherapy. Mechanism of action appears to be related to blockage of Fc receptors in the neonatal reticuloendothelial system. Studies have also documented decreased hemolysis after administration of IVIG using carboxyhemoglobin levels. Administration in doses of 500-1000 mg/kg in the first few hours of life to a newborn with severe hemolysis should be considered. However, efficacy depends on timing of administration, duration of treatment, and severity of hemolysis. Should be prepared by and dispensed from pharmacy and should not be mixed with normal saline. Dispensed as either 3% or 6% solution.

• References

Class Summary

These agents normalize antibody levels in patients with primary defective antibody synthesis. They prevent and treat certain bacterial and viral infections and reduce the immune-mediated hemolysis and phagocytosis.

• References

Epoetin alfa, recombinant (Epogen, Procrit)

• Dosing, Interactions, etc.

Clinical Context:  Purified glycoprotein produced from mammalian cells modified with gene coding for human erythropoietin (EPO). Amino acid sequence is identical to that of endogenous EPO. Biological activity mimics human urinary EPO, which stimulates division and differentiation of committed erythroid progenitor cells and induces release of reticulocytes from bone marrow into the blood stream.

• References

Class Summary

These agents may be required to correct anemia.

• References

Stannsoporfin (SnMP, Stanate)

Clinical Context:  Also known as tin-mesoporphyrin. Investigational in the United States. Phase III clinical trials completed. Structural analog of heme that blocks heme oxygenase (HO-1), a rate-limiting enzyme in bilirubin production, thereby preventing the conversion of heme to bilirubin. Heme is excreted unchanged in bile and is not stored in tissue. It is inert and does not enter the brain or interact with DNA. It does not affect previously formed bilirubin conjugation or excretion in liver. Several randomized, controlled and, when possible, blinded studies over the last decade that involved >700 neonates with all principle forms of neonatal jaundice have shown SnMP to be effective in preventing and blocking jaundice progression. Phototherapy was eliminated in 97% of treated infants.

Also inhibits nitric oxide synthase and soluble guanylyl cyclase. Repeated doses lead to inhibition of intestinal heme oxygenase involved in iron absorption and may lead to anemia. It also stimulates HO-1 transcription and protein levels. The half-life as measured in healthy adult volunteers is 3.8 h.

Available under the rules of a compassionate use protocol by WellSpring Pharmaceutical and InfaCare Pharmaceutical Corporations. For details, contact Dr Benjamin Levinson (732) 938-5885 ext 224, or by email at blevin@wellsringpharm.com.

• References

Class Summary

Phase III clinical trials have been completed in the United States on stannsoporfin, a competitive heme oxygenase inhibitor. The drug is currently available via a compassionate use protocol.

• References