Hemolytic Disease Of The Newborn and Neonatal Transfusion

Done by : Ala’a Abdullah Supervised by : Dr. Azmi Mahafzah

Introduction

• HDN is the destruction of the RBCs of the fetus and neonate by antibodies produced by the mother.

• The mother can be stimulated to form the antibodies by previous pregnancy or transfusion and sometimes during the second and third trimester of pregnancy.

• The incidence of the disease caused by anti-D has steadily decreased since 1968 with the introduction of Rh immune globulin (RhIg).

Over the years the disease has been known by various names describing the clinical symptoms:

1. hydrops fetalis : referring to the edema.2. erythroblastosis fetalis : referring to the nucleated red cells found in peripheral

blood.3. icterus gravis neonatorum : referring to the jaundice.

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• HDN is caused by the destruction of the RBCs of the fetus by antibodies produced by the mother.

• Only antibodies of the immunoglobulin G (IgG) class are actively transported across the placenta; other classes, such as IgA and IgM, are not.

• Most IgG antibodies are directed against bacterial, fungal, and viral antigens, so the transfer of IgG from the mother to the fetus is beneficial.

• However, in HDN, the antibodies are directed against those antigens on the fetal RBCs that were inherited from the father.

Etiology

• in Rh(D) HDFN, the Rh-positive firstborn infant of an Rh-negative mother is unaffected because the mother has not yet been immunized.

• During gestation, and particularly at delivery when the placenta separates from the uterus, variable numbers of fetal RBCs enter the maternal circulation.

• These fetal cells, carrying D antigen inherited from the father, immunize the mother and stimulate the production of anti-D.

Rh HDN

• Once the mother is immunized to D antigen, all subsequent offspring inheriting the D antigen will be affected.

• The maternal anti-D crosses the placenta and binds to the fetal Rh-positive cells.

• The sensitized RBCs are destroyed by the fetal reticuloendothelial system, resulting in anemia.

Factors Affecting Immunization and Severity

Antigenic Exposure• Fetomaternal hemorrhage during pregnancy can cause significant increases in

maternal antibody titers, leading to increasing severity of HDN.

• Transplacental hemorrhage of fetal RBCs into the maternal circulation occurs in up to 7 percent of women during gestation as determined by the acid-elution method for fetal hemoglobin (Kleihauer–Betke test)

• In addition, trauma to the abdomen, may increase the risk of fetomaternal hemorrhage.

• The volume of fetomaternal hemorrhage is small, 1mL of fetal RBCs can immunize the mother.

Kleihauer test, showing fetal red blood cells in rose-pink color, while adult red blood cells are only seen as "ghosts".

• The ability of individuals to produce antibody in response to antigenic exposure varies, depending on complex genetic factors.

• In Rh-negative individuals who are transfused with 200 mL of Rh-positive RBCs, about 85 percent form anti-D, and Nearly all the non responders will fail to produce anti-D even with repeated exposures to Rh-positive blood.

• the risk of immunization is only about 16 percent for an Rh-negative mother after an Rh-positive pregnancy if RhIg is not administered.

Host Factors

• Immunoglobulin class and subclass of the maternal antibody affect the severity of the HDN.

• Of all immunoglobulin classes, only IgG is transported across the placenta.

• The active transport of IgG begins in the second trimester and continues until birth.

• The IgG molecules are transported via the Fc portion of the antibodies.

• Of the four subclasses of IgG antibody, IgG 1 and IgG 3 are more efficient in RBC hemolysis,Therefore, the subclasses in the mother can affect the severity of the hemolytic disease.

Immunoglobulin Class

• Of all the RBC antigens, D is the most antigenic. For this reason, only Rh-negative blood is transfused to Rh-negative females of childbearing potential.

• Other antigens in the Rh system, such as C, E, and c, are also potent immunogens, These other Rh antibodies have been associated with moderate to severe cases of HDN.

• Of the non–Rh-system antibodies, anti-Kell is considered the most clinically significant in its ability to cause HDN.

Kell antigens are present on immature erythroid cells in the bone marrow, so severe anemia occurs not only from destruction of circulating RBCs but also from precursors.

Antibody Specificity

Influence of ABO Group• When the mother is ABO-incompatible with the fetus ,the incidence of detectable

fetomaternal hemorrhage is decreased.

• The ABO incompatibility protects somewhat against Rh immunization apparently by the hemolysis in the mother’s circulation of ABO-incompatible D-positive fetal RBCs before the D antigen can be recognized by the mother’s immune system.

Pathogenesis

Hemolysis, Anemia, and Erythropoiesis• Hemolysis occurs when maternal IgG attaches to specific antigens of the fetal RBCs. The

antibody coated cells are removed from the circulation by the macrophages of the spleen.

• Destruction of fetal RBCs and the resulting anemia stimulate the fetal bone marrow to produce RBCs at an accelerated rate, even to the point that immature RBCs (erythroblasts) are released into the circulation. The term “erythroblastosis fetalis” was used to describe this finding.

• When the bone marrow fails to produce enough RBCs to keep up with the rate of RBC destruction, erythropoiesis outside the bone marrow is increased in the hematopoietic tissues of the spleen and liver.

• The spleen and liver become enlarged (hepatosplenomegaly), resulting in portal hypertension and hepatocellular damage.

• The process of RBC destruction goes on even after such an infant is delivered alive, and as long as maternal antibody persists in the newborn infant’s circulation.

• IgG is distributed both extravascularly and intravascularly and has a half-life of 25 days, so antibody binding and hemolysis of RBCs continue for several days to weeks after delivery.

• Severe anemia along with hypoproteinemia caused by decreased hepatic production of plasma proteins leads to the development of high-output cardiac failure with generalized edema, effusions, and ascites, a condition known as hydrops fetalis.

• hydrops fetalis was almost uniformly fatal; currently, most fetuses with this condition can be treated successfully.

• The RBC destruction releases hemoglobin, which is metabolized to bilirubin. This bilirubin is called “indirect”.

• The indirect bilirubin is transported across the placenta and conjugated in the maternal liver to “direct” bilirubin. Then the conjugated bilirubin is excreted by the mother.

Bilirubin

• Although levels of total bilirubin in the fetal circulation and in the amniotic fluid may be elevated, these do not cause clinical disease in the fetus.

• after birth, accumulation of metabolic by-products of RBC destruction can become a severe problem for the newborn infant.

• The newborn liver is unable to conjugate bilirubin efficiently, especially in premature infants. With moderate to severe hemolysis, the unconjugated or indirect bilirubin can reach levels toxic to the infant’s brain and, if left untreated, can cause kernicterus (bilirubin encephalopathy) or permanent damage to parts of the brain.

Diagnosis and Management

• Serologic Clinical tests performed at appropriate times during the pregnancy can accurately determine the level of antibody in the maternal circulation, the potential of the antibody to cause HDN,and the severity of RBC destruction during gestation.

• If clinical and serologic data indicate that the fetus is becoming severely anemic, interventions such as intrauterine transfusion can be used to treat the anemia and prevent the development of severe disease.

• The recommended obstetric practice is to perform a type and-antibody screen at the first prenatal visit preferably during the first trimester.

• At that time, the pregnant woman can be asked about previous pregnancies and their outcomes and prior transfusions.

• antibody titers are useful in assessing the extent of intrauterine fetal anemia during the first affected pregnancy, antibody titers are less predictive in subsequent pregnancies.

Serologic Testing

• The testing of the specimen should include ABO and Rh testing for D antigen.

• Rh test may include weak D if no immediate reaction with anti-D occurs.

• The patient’s RBCs should also be tested simultaneously with Rh control reagent.

• testing for weak D to avoid false-positive reacions due to spontaneous agglutination of RBCs.

• If the patientis weak D–positive, the patient can be considered Rh-positive.

• In rare cases, weak D phenotype is caused by missing a part of the Rh Such patients may produce anti-D as an alloantibody, which has been reported to cause HDN.

ABO and Rh Testing

Antibody Screen• The test conditions must be able to detect clinically significant IgG alloantibodies that

are reactive at 37 C and in the antiglobulin phase.

• At least two separate reagent screening cells, covering all common blood group antigens.

• Antibody enhancing medium such as polyethylene glycol (PEG) or low ionic strength saline solution (LISS) can increase sensitivity of the assay.

• Many prenatal patients produce clinically insignificant antibodies, such as anti-Lea and/or anti-Leb.

• Therefore, immunohematology recommend omitting immediate spin and room temperature incubation phases and using anti-IgG rather than polyspecific antiglobulin reagent. These steps reduce detection of IgM antibodies, which cannot cross the placenta.

• If the antibody screen is reactive, the antibody identity must be determined.

• testing will depend on the antibody specificity.

• Cold reactive IgM antibodies such as anti-I, anti-IH, anti-Lea, anti-Leb, and anti-P1 can be ignored.

• Lewis system antibodies are rather common in pregnant women but have not been reported to cause HDN.

• Both anti-M and anti-N can cause mild to moderate HDN, although rarely.

• Other than anti-D, the most common and most significant antibodies are anti-K, anti-E, anti-c, anti-C, and anti-Fya.

Antibody Identification

Amniocyte Testing• If the mother has anti-D and the father is most likely to be heterozygous for the D

antigen.

• amniocentesis can be done as early as 10 to 12 weeks’ gestation to determine whether the amniocytes carry the gene for the D antigen.

• Amniocytes can be similarly tested for the genes coding c, e, C, E, K, Fya, Fyb,Jka, Jkb, and M.

• A specimen of the father’s blood should be obtained and tested for the presence and zygosity of the corresponding antigen.

• If the mother has anti-D and the father is D-positive, a complete Rh phenotype can help determine his chance of being homozygous or heterozygous for the D antigen by determining the most probable genotype.

• antibody specificity other than D, testing the father can save a great deal of time, expense, and worry if he is shown to lack the corresponding antigen.

• For example, only 10 percent of the random population is positive for the Kell antigen.

Paternal Phenotype

• The relative concentration of all antibodies capable of crossing the placenta and causing HDN must be determined by antibody titration.

• The patient serum is serially diluted and tested against appropriate RBCs to determine the highest dilution at which a reaction occurs.

• The method must include the indirect antiglobulin phase using anti-IgG reagent. The result is expressed as either the reciprocal of the titration end-point or as a titer score.

• The titration must be performed exactly the same way each time the patient’s serum is tested.

• The RBCs used for each titration should have the same genotype approximately the same storage time, and the same concentration.

Antibody Titers

• In general, a titer of 16 to 32 is considered significant. If the initial titer is 16 or higher, a second titer should be done at about 18 to 20 weeks’ gestation. A titer reproducibly and repeatedly at 32 or above represents an indication for amniocentesis or percutaneous umbilical blood sampling between 18 and 24 weeks’ gestation.

• When the titer is less than 32, the titer should be repeated at 4-week intervals, beginning at 18 to 20 weeks’ gestation, and every 2–4 weeks during the third trimester. The last determination should be made within a week of the expected date of delivery.

• Antibody titer alone cannot predict severity of HDN, In some sensitized women, the antibody titer may remain moderately high throughout pregnancy while the fetus is becoming more severely affected.

• previously sensitized woman may have consistently high antibody titer whether pregnant or not and, if pregnant, whether the fetus is Rh-positive or Rh-negative, In others, the titer may rise rapidly, which portends increasing severity of HDN.

• Patients with a history of a severely affected fetus or early fetal death may require earlier intervention.

• Under ultrasound guidance, amniocentesis is done to assess the status of the fetus.

• The concentration of bilirubin pigment in the amniotic fluid correlates with the extent of fetal anemia.

• The amniotic fluid is subjected to a spectrophotometric scan at steadily increasing wavelengths, so that the change in the optical density (▲OD) at 450 nm (the absorbance of bilirubin) can be calculated.

• The measurement is plotted on the Liley graph according to gestational age.

• An increasing or unchanging ▲OD 450 nm as pregnancy proceeds predicts worsening of the fetal hemolytic disease and the need for frequent monitoring and intervention if indicated.

Amniocentesis and cordocentesis

• Values in zone III indicate severe and often life threatening hemolysis (fetal hemoglobin less than 8 g/dL) and require urgent intervention.

• In zone II, most fetuses have moderate disease that may require intervention.• Values in zone I predict mild or no disease, which do not require intervention.

• Recent advances in sonography have allowed clinicians to obtain a sample of the fetal blood through a procedure called percutaneous umbilical blood sampling, or cordocentesis.

• Using high-resolution ultrasound with color Doppler enhancement of blood flow, the umbilical vein is visualized at the level of the cord insertion into the placenta.

• A needle is inserted into the umbilical vessel, and a sample of the fetal blood is obtained. The fetal blood sample can then be tested for hemoglobin, hematocrit, blood type, antigen phenotype, and DAT.

• Intrauterine transfusion becomes necessary when one or more of the following conditions exists:

1. Amniotic fluid ▲OD 450 nm results are in high zone II or in zone III.2. Cordocentesis blood sample has hemoglobin level less than 10 g/dL.3. Fetal hydrops is noted on ultrasound examination.

• Intrauterine transfusion can be performed by injecting the RBCs into the fetal peritoneal cavity where the RBCs can be absorbed into the circulation.

• More recently, cordocentesis has been used to inject donor RBCs directly into the fetal umbilical vein.

• The goal of intrauterine transfusion is to maintain fetal hemoglobin above 10 g/dL.

Intrauterine Transfusion

• Once intrauterine transfusion is initiated, the procedure is repeated every 2 to 4 weeks until 34 to 36 weeks’ gestation or until the fetal lungs are mature, when early delivery can be performed.

• The initial intrauterine transfusion is rarely performed after 34 weeks’ gestation.

• Intrauterine transfusion can suppress the fetal bone marrow from producing RBCs.

• During the first weeks after birth, the infant may require RBC transfusion.

Early Delivery

• Early delivery was used for many years for moderate-to-severe disease to interrupt the transport of maternal antibody to the fetus and to allow exchange transfusion.

• With the use of repeated and frequent IV transfusions in the fetus, delivery before the lungs are mature can usually be avoided.

• after delivery, phototherapy with ultraviolet light can be used to change unconjugated bilirubin to biliverdin.

• In infants with mild-to-moderate hemolysis, the use of phototherapy may avoid the need for exchange transfusion to treat hyperbilirubinemia and anemia.

Phototherapy

• The therapy uses a blue light (420-470 nm) that converts bilirubin so that it can be excreted in the urine and feces.

• Soft eye shields are placed on the baby to protect their eyes from damage that may lead to retinopathy due to the lights.

Serologic Testing of the Newborn

ABO Grouping • ABO antigens are not fully developed in newborn infants and thus may give weaker

reactions than in older children and adults.

• the infant does not have his or her own isoagglutinins but may have those of the mother, so reverse grouping cannot be used to confirm the ABO group.

Rh Typing • Rarely, the infant’s RBCs can be heavily antibody-bound with maternal anti-D

causing a false-negative Rh type, or what has been called blocked Rh.

• An eluate from these RBCs will reveal anti-D, and typing of the eluted RBCs will show reaction with anti-D.

DAT• The most important serologic test for diagnosis of HDN is the DAT with anti-IgG

reagent. The positive test result indicates antibody is coating the infant’s RBCs; however, the strength of the reaction does not correlate well with the severity of the HDN.

• A positive test result may be found in infants without clinical or other laboratory evidence of hemolysis (e.g., mother received RhIg).

Elution• The routine preparation of an eluate of all infants with a positive DAT result is

unnecessary.

• Elution in cases of known HDN and postnatal ABO incompatibility is not needed, because eluate results do not change therapy.

Newborn Transfusions

• The newborn infant may receive small aliquot transfusions or exchange transfusions, or both. Small aliquots can be used to correct anemia when the bilirubin level is not high enough to warrant an exchange transfusion.

• Exchange transfusions are used primarily to remove high levels of unconjugated bilirubin and thus to prevent kernicterus.

• Other advantages of exchange transfusion include the removal of part of the circulating maternal antibody, removal of sensitized RBCs, replacement of incompatible RBCs with compatible RBCs, and suppression of erythropoiesis.

• Premature newborn infants are more likely than full-term infants to require exchange transfusions for elevated bilirubin because their livers are less able to conjugate bilirubin.

• full-term newborn infants normally have rather high hemoglobin levels (14 to 20 g/dL), a level below 12 g/dL is considered anemia that may require transfusion.

• Lower than 8 g/dL is considered severe anemia and corresponds to zone III of the Liley graph.

• 8 to 12 g/dL corresponds to zone II.

Testing Baby• Antibody elution testing from cord red blood cells.

• ABO/D typing– If baby received intrauterine transfusions will type as O negative– If baby’s Direct Antiglobulin Test is strongly positive due to anti-

D may get false negative immediate spin reaction with reagent anti-D (blocking phenomenon), weak D (Du) test will be strongly positive.

• Antibody screen.

• Coomb’s crossmatch antigen negative donor.

Testing Mom

• Type and screen on mom.

• Identification of unexpected antibodies.

• More than 40 antigens have been identified as causing HDN.

• Select blood that lacks antigens to which mom has antibodies.

• Perform coomb’s crossmatch with Mom and baby’s blood.

Selection of Blood• Most centers treating HDN use group O RBCs for intrauterine as well as neonatal

transfusions.

• The RBCs must be antigen-negative for the respective antibodies.

• Rh-negative units are selected for fetuses and neonates whose blood types are unknown or are Rh-negative.

• For intrauterine transfusion, the hematocrit level of the RBCs should be greater than 70 percent, because of the small volume transfused and the need to correct severe anemia.

• For exchange transfusions, one practice is to prepare RBCs from whole blood units and then replace the plasma with group AB plasma to reduce the amount of blood group antibodies transfused.

• Blood transfused to the fetus and premature infant should also be irradiated to prevent graft-versus-host disease.

• blood for exchange transfusion not contain hemoglobin S.

• CPD, as fresh as possible, preferably <5 days old.

• A hematocrit of 70% or greater is desirable to minimize the chance of volume overload in the fetus.

• The volume transfused ranges from 75-175 mL depending on the fetal size and age.

• CMV negative , Hemoglobin S negative.

• Irradiated.

• O negative, lack all antigens to which mom has antibodies and Coomb’s compatible.

Beneficial Effects of Exchange Transfusion

• Removal of bilirubin.• Removal of sensitized RBCs.• Removal of incompatible antibody.• Replacement of incompatible RBCs with compatible RBCs.• Suppression of erythropoiesis (reduced production of incompatible

RBCs).

RhIg

• Active immunization induced by RBC antigen can be prevented by the concurrent administration of the corresponding RBC antibody. This principle has been used to prevent immunization to D antigen by the use of high-titered RhIg.

• During pregnancy and delivery, mixing of fetal and maternal blood occurs. If the mother is Rh-negative and the fetus is Rh-positive, the mother has up to a 9 percent chance of being stimulated to form anti-D.

• Before delivery, the risk of sensitization is 1.5 to 1.9 percent of susceptible women, indicating that a significant amount of fetal RBCs can enter the maternal circulation during pregnancy.

• the greatest risk of immunization to Rh is at delivery.

Mechanism of Action

• The administered RhIg attaches to the fetal Rh-positive RBCs in the maternal circulation.

• The antibody-coated RBCs are removed by the macrophages in the maternal spleen.

• The RBC antigens are thus unavailable for dendritic cells to present antigen to T helper cells.

• The amount of antibody necessary to prevent alloimmunization has been determined experimentally and is known to be less than that required to saturate all D antigen sites.

Indications

Antenatal • Because of the known risk of Rh immunization during pregnancy, RhIg should be

given early in the third trimester, or at about 28 weeks’ gestation.

• The dose does not pose a risk to the fetus, inasmuch as this amount will cause a titer of only 1 or 2 in the mother.

• positive DAT result may be observed in the newborn.

Postpartum

• The Rh-negative nonimmunized mother should receive RhIg soon after delivery of an Rh-positive infant

• recommended interval is within 72 hours after delivery

• if more than 72 hours have elapsed, RhIg should still be given, inasmuch as it may be effective and is not contraindicated.

• The mother should be D-negative. the infant should be D-positive or weak D-positive. If the type of the infant is unknown (e.g., if the infant is stillborn), RhIg should also be administered.

• It is essential that the anti-D from antenatal RhIg present at delivery not be interpreted erroneously as active rather than passive immunization.

• Omission of the indicated dose after delivery may lead to active immunization.

Additional Indications for RhIg• Amniocentesis• Chorionic villus sampling• Abortion (spontaneous and induced)• Ectopic pregnancy• Abdominal trauma• Accidental or inadvertent transfusion• Greater than 40 weeks gestation

ABO HDN

• ABO incompatibility between the mother and newborn infant can cause HDN. Maternal ABO antibodies that are IgG can cross the placenta and attach to the ABO-incompatible antigens of the fetal RBCs.

• Destruction of fetal RBCs leading to severe anemia is extremely rare, More commonly, the disease is manifested by the onset of hyperbilirubinemia and jaundice within 12 to 48 hours of birth.

• The increasing levels of bilirubin can be treated with phototherapy Severe cases requiring exchange transfusion are extremely rare.

• ABO incompatibility has become the most common cause of HDN. Statistically, mother and infant are ABO-incompatible in one of every five pregnancies

Factors Affecting Incidence and Severity• ABO antibodies are present in the sera of all individuals whose RBCs lack the corresponding

antigen.

• These antibodies, the result of environmental stimulus, occur more frequently as high-titered IgG antibodies in group O individuals than in group A or B individuals. Most occur in group A infants in white populations. black population group B infants are more often affected.

• The mother’s history of prior transfusions or pregnancies seems unrelated to the occurrence and severity of the disease.

• tetanus toxoid administration and helminth parasite infection during pregnancy have been linked to the production of high-titered IgG ABO antibodies and severe HDN.

• high-titered IgG antibodies that are transported across the placenta seem incapable of causing significant RBC destruction in an ABO-incompatible fetus. These infants are delivered with mild anemia or normal hemoglobin levels.

• The mild course of ABO HDN is related more to the poor development of ABO antigens on fetal RBCs than the characteristics of the maternal antibody.

• ABO antigens are not fully developed until after the first year of life.

• The laboratory findings in ABO HDN differ from those for Rh disease. Microspherocytes and increased RBC fragility in the infant are characteristic of ABO HDN but not of Rh HDN.

• The serious consequences of Rh and other blood groups causing HDN, such as stillbirth, hydrops fetalis, and kernicterus, are extremely rare in ABO HDN.

Prenatal Screening

• Many workers have tried to use the immunoglobulin class and titer of maternal ABO antibodies to predict ABO HDN. These tests are laborious and at best demonstrate the presence of IgG maternal antibody but do not correlate well with the extent of fetal RBC destruction.

• detection of ABO HDN is best done after birth.

• No single serologic test is diagnostic for ABO HDN. • When a newborn infant develops jaundice within 12 to 48 hours after birth, various

causes of jaundice need to be investigated, and ABO HDN is only one.

• The DAT on the cord or neonatal RBCs is the most important diagnostic test. In all cases of ABO HDN requiring transfusion therapy, the DAT result has been positive.

• Collecting cord blood samples on all delivered infants is highly recommended.

• In the rare cases in which ABO incompatibility can be the only cause of neonatal jaundice but the DAT result is negative, the eluate of the cord RBCs always reveals ABO antibodies. The eluate can also be helpful when the mother’s blood specimen is not available.

Postnatal Diagnosis