Hemolytic Disease of The Fetus and Newborn

HDFN

• aka erythroblastosis fetalis

• the destruction of the RBCs of a fetus and/or neonate by IgG antibodies produced by the mother.

• ~95% of the cases were caused by antibodies in the mother directed against the Rh antigen D

• incidence rate for anti-D has decreased since intro of Rh immune globulin (RHIG)

• Rh(D) incompatibility is common although others exist.

What is the etiology of HDFN?

• RBCs of a fetus are destroyed by antibodies produced by mother’s immune system.

• only IgG antibodies are actively transported across the placenta. These antibodies, directed against fetal antigens, cross the placenta, sensitize the fetal red cells and shorten the red cell survival. This premature red cell destruction can cause mild anemia to death in utero.

• The antibodies are directed against those antigens on the fetal RBCs that were inherited from the father.

Rh HDFN prevalence

• the Rh positive first born infant of an Rh negative mother usually is unaffected because the mother has not yet been immunized.

• During gestation and delivery, then the placenta separates from the uterus, fetal RBCs enter the maternal circulation.

• When D antigen is inherited from the father, these fetal cells immunize mother and stimulate the production of anti-D.

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

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

ABO HDFN

• 20% of pregnancies are ABO incompatible.

• Maternal ABO antibodies (isohemagglutinins) that are IgG can cross the placenta and attach to the ABO antigens of the fetal RBCs.

• ABO HDFN is nearly always limited to A or B infants of group O mothers with potent anti-A,B antibodies.

• Mild anemia course is related due to the poor development of ABO antigens on fetal RBCs.

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

Lab results that indicate ABO HDFN?

• Microspherocytes and increased RBC fragility.

• Bilirubin peak at 1 to 3 days

• Severity of the disease does not correlate to the presence of a positive DAT.

Treatment of ABO HDFN

• Phototherapy is usually sufficient for slowly rising bilirubin levels.

• Rarely a need for exchange transfusion.

• Extremely rare occurrences of stillbirth, hydrops fetalis, and kernicterus.

Antigenic Exposure

• Transplacental hemorrhage of fetal RBCs into the maternal circulation occurs in up to 7% of women during gestation.

• At delivery, the incidence is more than 50%

• Interventions such as amniocentesis and chorionic virus sampling, as well as trauma to the abdomen can increase the risk of fetomaternal hemorrhage.

The ability of individuals to produce antibodies in response to antigenic exposure varies, depending on complex genetic factors:

• Of the four subclasses of IgG antibody, IgG1 and IgG3 are more efficient in RBC hemolysis than are IgG2 and IgG4.

• The subclass(es) in the mother can affect the severity of the hemolytic disease.

Of all RBCs antigens, which is the most antigenic?

• antigen D

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

Influence of ABO group

• When the mother is ABO-incompatible with the fetus (major incompatibility), the incidence of detectable fetomaternal hemorrhage is decreased.

• The ABO incompatibility can protect 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.

Hemolysis, Anemia, and Erythropoiesis

• Hemolysis occurs when maternal IgG attaches to specific antigens of the fetal RBCs.

• Rate of fetal RBC destruction depends on antibody titer, specificity, and number of RBC antigenic sites.

• Fetal RBC destruction and the resulting anemia stimulate the fetal bone marrow to produce RBCs at an accelerated rate.

• Immature fetal RBCs (erythroblasts) are released into the circulation: “erythroblastosis fetalis.”

• Hepatosplenomegaly results from extramedullary hematopoiesis.

• Severe anemia and hypoproteinemia lead a condition known as “hydrops fetalis.”

• The rate of RBC destruction after birth decreases because no additional maternal antibody is entering the infant’s circulation through the placenta.

• Effect of half life of maternal IgG

Hydrops fetalis

• the development of high-output cardiac failure with generalized edema, effusions, and ascites caused by severe anemia and hypoproteinemia

How does Kernicterus happen?

• RBC destruction releases hemoglobin, which is metabolized to bilirubin.

• Maternal liver metabolizes unconjugated bilirubin.

• After birth, the immature newborn liver cannot adequately process unconjugated bilirubin.

• With moderate to severe hemolysis, the unconjugated, or indirect, bilirubin can reach levels toxic to the infant’s brain (generally, more than 18 to 20 mg/dL)

Postnatal Diagnosis

• Development of jaundice within 12 to 48 hours after birth

• Positive DAT (cord or neonatal RBCs)

• Negative DAT with infant jaundice requires other causes of jaundice should be investigated

• May require elution studies of cord blood

Actions taken to prevent HDFN

• Type and antibody detection test should be performed during the first trimester.

• Maternal history must be taken

  • history of HDFN

  • previous pregnancy outcomes

  • history prior transfusions.

• Previous severe disease and poor outcome predict similar findings in the current pregnancy.

Antibody Identification

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

• Follow-up testing will depend on the antibody specificity and likely clinical significance.

• Many Rh-negative pregnant women have weakly reactive anti-D, particularly at the third trimester.

• Most of these women have received RhIG, either after an event with increased risk of fetomaternal hemorrhage or at 28 weeks’ gestation (antenatal).

• The passively administered anti-D will be weakly reactive in testing and will remain demonstrable for 2 months or longer.

• This must be distinguished from active immunization.

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

Antibody Titers

• The relative concentration of all antibodies capable of crossing the placenta and causing HDFN is determined by antibody titration.

  • The method must include the indirect antiglobulin phase using anti-IgG reagent.

  • The result is expressed as either the reciprocal of the titration endpoint or as a titer score.

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

• A titer reproducibly and repeatedly at 32 or above represents an indication for Color Doppler Middle Cerebral Artery Peak Systolic Velocity studies after 16 weeks’ gestation for determination of the presence of fetal anemia.

Color Doppler Middle Cerebral Artery-Peak Systolic Velocity (MCA-PSV)

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

• The measurement of the fetal MCA-PSV with color Doppler ultrasonography can reliably predict anemia in the fetus.

Paternal Phenotype and Genotype

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

• A more sensitive and precise genotype can be determined by DNA methods.

• The information guides further testing of the mother and counseling for potential treatment plans and complications of HDFN.

Fetal DNA Testing?

• If the mother has anti-D and the father is likely to be heterozygous for the D antigen, amniocentesis or chorionic villous sampling can be performed as early as 10 to 12 weeks’ gestation to determine whether the fetus has the gene for the D antigen.

• Testing can be performed for the genes coding c, e, C, E, K, Fya, Fyb, Jka, Jkb, M and others.

Management of the fetus

• Ultrasound

• Cordocentesis

• Amniocentesis

  • The concentration of bilirubin pigment in the amniotic fluid measured by the ∆ABS 450 nm procedure as pregnancy proceeds predicts worsening of the fetal hemolytic disease.

Intervention in the form of intrauterine transfusion becomes necessary when one or more of the following conditions exists:

• • MCA-PSV indicates anemia.

  • Fetal hydrops is noted on ultrasound examination.

  • Fetal hemoglobin level is less than 10 g/dL.

  • Amniotic fluid ∆OD 450 nm results are high.

    • Risks and benefits of intrauterine transfusion must be weighed and evaluated.

Management of the infant

• Cord Blood Testing

  • ABO Grouping

  • RhD Typing

  • Direct Antiglobulin Test

  • Elution

Use of Whole Blood or equivalent

• used to replace the neonate’s circulating blood.

• Used primarily to remove high levels of unconjugated bilirubin

• Other advantages: removal of part of the circulating maternal antibody, removal of sensitized RBCs, and replacement of incompatible RBCs with compatible RBCs

Serologic testing of the cord cells

• used to confirm HDFN and prepare for possible transfusion.

• ABO grouping (ABO antigens are not fully developed in newborns and may show weaker reactions)

• Rh typing

• Direct Antiglobulin Test (DAT)

• Elution

Phototherapy

• After delivery, the neonate can develop hyperbilirubinemia of unconjugated bilirubin.

• Phototherapy at 460 to 490 nm is used to change the unconjugated bilirubin to isomers, which are less lipophilic and less toxic to the brain.

Intravenous immune globulin (IVIG)

• The IVIG competes with the mother’s antibodies for the Fc receptors on the macrophages in the infant’s spleen, reducing the amount of hemolysis.

Selection of Blood for Intrauterine and Neonatal transfusion

• Most centers treating HDFN use group O RBCs for intrauterine as well as neonatal transfusions.

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

• Donors are usually cytomegalovirus (CMV)-negative as well.

• Traditionally, blood units less than 7 days from collection from the donor are selected.

Rh Immune Globulin

• 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.

• If the mother is Rh-negative and the fetus is Rh-positive, the mother has up to a 16% chance of being stimulated to form anti-D.

What are the two maternal assays for detecting FMH?

• Kleihauer-Betke test and flow cytometry

• Both are based on identification of hemoglobin F, the predominant fetal hemoglobin.

• Both assays measure the volume of fetal blood in the maternal circulation at a point in time and thus do not necessarily indicate the volume of blood loss over time, if bleeding was chronic or occurred on multiple occasions.

Kleihauer-Betke test

• the main diagnostic test for detection and quantitation of FMH .Red blood cells from the maternal circulation are fixed to a slide that is exposed to an acidic pH solution.

• Adult red blood cells become "ghost" cells since hemoglobin A is soluble and eluted across membrane defects at a low pH. Fetal red blood cells remain pink because hemoglobin F is stable at pHs in this range.

• The volume of fetal whole blood (mL) in the maternal circulation is: (% fetal cells) X (maternal hematocrit [%] divided by fetal hematocrit [%]) X (maternal blood volume( mL).

Flow cytometry

• another assay for detecting and quantitating FMH.

  A monoclonal antibody to hemoglobin F is conjugated to a fluorochrome and used to detect fetal hemoglobin in permeabilized cells as they pass through the channel of a flow cytometer.

Flow cytometry Vs Kleihauer-Betke

• flow cytometry is more accurate, more reproducible, and less labor intensive

Rh immune Globulin mechanism of action

• The mechanism of action is uncertain.

• Evidence indicates RhIG interferes with B-cell priming to make anti-D, although other modes of action may occur.

• The Rh-negative nonimmunized mother should receive RhIG within 72 hours after delivery of an Rh-positive infant

Dose and Administration

• The regular-dose vial of RhIG in the United States contains sufficient anti-D to protect against 15 mL of packed RBCs or 30 mL of whole blood.

• Use of Kleihauer-Betke test to determine volume of fetal hemorrhage.

Maternal Weak D

• In certain patients, serologic reagents do not accurately detect the RhD type.

• RhD genetic testing for patients with a weak D phenotype may provide accurate and actionable results for RhD blood typing and RhIG administration

RHIG facts

• RhIG is of no benefit once a person has been actively immunized and has formed anti-D

• RhIG is not indicated for the mother if the infant is found to be D-negative.

When Fetal anemia is suspected…

• the peak systolic velocity (PSV) of the middle cerebral artery (MCA) should be determined with Doppler ultrasound.

• A MCA-PSV value ≥1.5 multiples of the median (MoM) strongly correlates with moderate to severe fetal anemia

• Either a Kleihauer-Betke stain or flow cytometry can be used for diagnosing FMH.

• The volume of fetal blood loss should be calculated as a percentage of the estimated fetoplacental blood volume.