Chapter 12: Hemolytic Disease of the Fetus and Newborn

What is HDFN also known as?

Erythroblastosis fetalis

What is HDFN?

Fetal or newborn red blood cells (RBCs) are destroyed by maternal IgG Antibodies

Maternal antibodies:

Cross the placenta

Sensitize fetal RBCs

Shorten RBC survival

How does HDFN occur? (*In order of most to least common)

Fetal cells escape into the maternal circulation as a result of a fetomaternal hemorrhage via:

• Delivery

• Amniocentesis

• Abortion (spontaneous or induced)

• Cordocentesis

• Ectopic pregnancy

• Abdominal trauma

Fetal RBC antigens that the mother does not have may stimulate the mother to produce antibodies

Antibodies bind to fetal antigens and cause RBC destruction in the fetal liver and spleen

What happens during HDFN before birth?

Bilirubin crosses through the placenta and is conjugated by the maternal liver

As RBC destruction continues, fetal erythropoiesis increases:

• Erythroblasts are released (erythroblastosis fetalis), spleen and liver may enlarge

• Edema occurs in the peritoneal and pleural cavities (hydrops fetalis)

Cardiac failure may result

What happens during HDFN after birth?

Newborn cannot conjugate bilirubin

Unconjugated bilirubin binds to albumin and then to tissues, causing jaundice

Permanent brain damage (kernicterus) may result if bilirubin binds to tissues of the CNS

What 3 factors must be present for HDFN to occur?

1. RBC antibody must be IgG - Only IgG crosses the placenta

2. Fetus must possess an antigen that the mother lacks - (the gene is inherited from the father)

3. Antigen must be well developed at birth - (Lewis, Lutheran, I, IH, P1 not well developed)

What are the three types of HDFN?

Rh (D antigen)

ABO

Other Antibodies

What is the most severe type of HDFN?

Rh HDFN

What is Rh HDFN?

D-negative women are sensitized during the first pregnancy with a D-positive baby (without intervention)

Subsequent pregnancies are affected

Infant results:

• Positive direct antiglobulin test (DAT)

• Jaundice and/or anemia may occur

Exchange transfusion may be necessary

Does Rh HDFN have a positive or negative DAT?

Positive

How can Rh HDFN be prevented?

Rh immune globulin (RhIG) is given to prevent Rh HDFN

How does RhoGAM work?

• RhoGAM anti-D attaches to fetal D positive cells

• prevents maternal antibody from recognizing D positive cells

What is the initial dose of RhIG given to mothers and what protection does it provide?

• 300 ug given at 28 weeks gestation

• protection for up to 15 mL of D positive fetal RBCs OR 30 mL of fetal whole blood

When is RhIG is given to postpartum mothers?

• one full dose within 72 hours of delivery

• more than one dose may be necessary if the mother has a fetomaternal hemorrhage of more than 30 mL of blood

What is the most common type of HDFN?

ABO HDFN

What is ABO HDFN?

Mother has group O blood; baby has group A or B blood

First pregnancy may be affected (Antibodies already present)

Production of mild symptoms is possibly due to:

• A or B substances in tissue that may neutralize antibodies

• Fetal/infant RBCs may be poorly developed

• Fetal/infant RBC sites may be reduced

Some jaundice may occur

Phototherapy can be used to treat jaundice

Other types of HDFN

Any IgG can cause HDFN

Anti-c and anti-K antibodies are common causes

Other Kell antibodies and antibodies to Kidd, Duffy, S, and U antigens are less common

Agglutination with paternal cells and maternal serum is a clue to a low-frequency antigen

• selected cells can be used to determine the antibody identity

What two purposes does prenatal type and screen testing serve?

Perform a type and screen on pregnant females:

1. Identifies D-negative women who are candidates for RhIG

2. Identifies women with antibodies capable of causing HDFN

What is antibody titration and why is it helpful?

procedure that measures the strength of an antibody by testing its reactivity at increasing dilutions against its corresponding antigen

Titration helps determine whether certain procedures should be performed

Antibody titration procedure

1. patient serum is added to tube 1

2. an equal amount of saline is added to the rest of the tubes

3. 0.5 mL of patient serum is removed from the first tube and added to the tube 2

4. 0.5 mL are removed from tube 2 and added to tube 3 (so on and so forth)

5. incubate and add a drop of AHG reagent

6. observe for agglutination (the last reacting tube is the titer, if tube 3 is positive the titer is 1:8 or 8)

if you are trying to detect anti-K (for example) you would need to add one drop of Kell homozygous screening cell to each tube for agglutination

Antibody Titration result interpretations

Baseline titer is determined in the first trimester and repeated at 4- to 6-week intervals (sample is frozen for future testing)

• A titer that rises by 2 dilutions (compared to baseline) is significant

• A titer of 16 or 32 is usually critical for anti-D and other Rh antibodies

How can an Ultrasound detect fetal anemia?

Color Doppler ultrasonography can detect fetal anemia

• Increased cardiac output and low blood viscosity are indicative of anemia

Severity of anemia is determined by evaluating the peak systolic velocity

non-invasive

How is amniocentesis performed?

Amniotic fluid is scanned spectrophotometrically for 350 to 700 nm

The optical density is plotted on a Liley graph (using gestational age) into 3 zones:

• Upper zone (zone 3): severe HDFN

• Middle zone (zone 2): moderate disease

• Lower zone (zone 1): mild disease

How is fetal hemolysis determined with amniotic fluid?

by measuring the concentration (optical density) of bilirubin

What are the three courses of action that are taken based on amniocentesis results?

1. Pregnancy continues to term

2. Intrauterine transfusion is performed

3. Early labor is induced

What is Cordocentesis?

Fetal blood sample is taken for:

• Hemoglobin and hematocrit

• Bilirubin testing

• RBC genotyping

Cordocentesis can be used for intravascular transfusions in cases of severe HDFN

What is Fetal Genotyping?

maternal plasma is typed for fetal DNA using molecular techniques during the second trimester

Predicting fetal genotype can:

• avoid invasive procedures like amniocentesis or cordocentesis

• help predict if the fetus has an antigen that can cause HDFN

What is postpartum D testing of newborns?

All infants born to D-negative mothers are D tested, including weak D antigen testing

False-negative results:

• D-antigen sites are blocked by antibody

  • Perform elution (will demonstrate anti-D antibody)

False-positive results:

• occurs if a weak D test is performed on RBCs coated with some other antibody (indicated by a positive DAT)

Postpartum ABO testing of newborns

only forward typing is performed

cord blood is washed with saline to get rid of Wharton’s jelly (to prevent false-positive agglutination)

ABO HDFN should be suspected if:

• the mom is group O with a negative antibody screen

• the infant is group A, B, or AB with a positive DAT

Elution may be necessary for ABO HDFN if:

the baby’s DAT is positive and if the mother's antibody has not been identified or the maternal serum sample is unavailable

If the eluate is negative, an antibody to low-frequency antigen is suspected

If the eluate is positive with A or B cells and negative with screening cells, ABO HDFN is indicated

What are Intrauterine Transfusions?

• transfusions performed to correct fetal anemia or heart failure

• done today by cordocentesis

  • direct vascular access

  • benefits: ability to obtain blood samples for typing, DAT, antigen typing, H&H, and bilirubin

What blood should be used for intrauterine transfusions?

• group O, D negative

• fresh (collected within 7 days prior to transfusion)

• irradiated (to prevent GVHD)

• CMV negative and/or leukoreduced

• negative for Hgb S

• have a hematocrit of 75% to 80% (to avoid volume overload)

• antigen negative if the mother has any alloantibody

Who are screened for fetomaternal hemorrhages?

RhIG candidates

What is the most common screening method for fetomaternal hemorrhages?

Rosette Test

• a postpartum maternal specimen is collected 24 hours after delivery to allow for enough time for fetal cells to circulate if hemorrhage did occur

How do you do a Rosette Test?

1. a 3-4% suspension is made with the mother’s RBCs

2. one drop of cell suspension is added with a specific anti-D reagent

3. incubate (anti-D binds to any fetal D pos cells)

4. wash

5. add D positive indicator cells (binds to anti-D and forms a rosette pattern with the D pos fetal cells)

6. suspension is observed under the microscope

When observing the rosette test under the microscope, how do you interpret the results?

• less than 1 rosette per 3 low-power fields: one vial of RhIG is given to the mother

• more than 1 rosette per 2 low-power fields: the bleed must be quantitated for the appropriate dose of RhIG

Causes of false positives and false negatives in the rosette test

• false positives

  • occurs if the mother is weak D positive (shows false agglutination)

• false negatives

  • occurs if the fetus is weak D positive (D antigen expression is too weak to bind to anti-D)

How do you quantify fetomaternal hemorrhage?

• flow cytometry

  • measures fetal Hgb or D positive red cells

• Kleihauer-Betke test

How do you do the Kleihauer-Betke Test?

1. Make blood smear with the mother’s postpartum specimen

2. Expose to acid buffer

3. Wash

4. Stain

5. Examine under microscope

fetal Hgb is resistant to the acid, so they appear dark pink

adult Hgb is not resistant, so they are pale pink

**Kleihauer-Betke Calculation**

IN NOTES

What is Phototherapy?

Initial treatment for hyperbilirubinemia and for mild cases of HDFN (ABO HDFN)

Uses fluorescent blue light (420 to 475 nm)

Light converts bilirubin to isomers that are excreted in the bile

If patient is unresponsive, exchange transfusion may be necessary

What is Exchange Transfusion?

Replacement of a newborn’s blood volume to treat HDFN

What does an Exchange Transfusion do?

Corrects anemia without expanding the blood volume

Removes newborn's RBCs and replaces them with antigen-negative cells

Reduces bilirubin (prevents kernicterus)

Reduces maternal antibody in the baby’s blood

How is Exchange Transfusion performed?

an ABO and D typing is determined in the infant

Maternal or infant serum or plasma is used for antibody screening

Antigen-negative, AHG crossmatch compatible units are given if antibody is present (O neg is usually given)

When is RhIG given?

• amniocentesis

• spontaneous or induced abortions

• ectopic pregnancies

• abdominal trauma

• during transfusions

• those who are over 40 weeks pregnant

What must be done to the blood when a baby is receiving an exchange transfusion?

• must be reconstituted with fresh frozen plasma to a HCT between 45% - 60%

• CMV negative

• Hgb S negative

Why is phototherapy used to treat HDFN?

used to treat hyperbilirubinemia

What is the greatest danger to a fetus affected by HDFN before delivery?

• kernicterus

• anemia

• hyperbilirubinemia

• hypotension

amemia

What volume of whole blood is covered in a fetomaternal hemorrhage with a 300 ug dose of RhIG?

• 10 mL

• 15 mL

• 30 mL

• 50 mL

30 mL

What is the greatest danger to a newborn affected by HDFN postpartum?

• kernicterus

• anemia

• conjugated bilirubin

• immature lung development

kernicterus

baby cannot get rid of excess bilirubin so it builds up

Which antibody shows no risk of HDFN?

• anti-Le^a

• anti-C

• anti-K

• anti-S

anti-Le^a

What requirement is important when selecting blood for exchange transfusion to avoid elevated levels of potassium?

blood less than 7 days old

A mother is group A, D negative with anti-D in her plasma. Select the appropriate unit for an intrauterine transfusion

• group O neg

• group O pos

• group A neg

• group A pos

group O neg

Select the statement that is true regarding the rosette test.

• performed on a cord blood sample

• used to screen for FMH

• a quantitative test used to calculate the volume of FMH

• an acid elution used to estimate the volume of FMH

used to screen for FMH

Which test is not necessary when testing a cord blood sample?

• ABO

• D

• DAT

• antibody screen

antibody screen

A group A, D negative mother demonstrating anti-D antibodies delivered a group O negative baby with a positive DAT, elevated bilirubin, and low Hgb. Which is the most probable explanation for these test results?

• ABO HDFN

• HDFN with a false-negative D type due to blocking of antibodies

• large FMH causing discrepancy in blood type

• prenatal RhIG administration

HDFN with a false-negative D type due to blocking of antibodies

What amount of FMH bleed is detectable by the rosette test?

10 mL

A rosette test performed on a D negative mother who delivered a D positive baby demonstrated five total rosettes in five fields observed. What is the correct course of action?

• submit the sample for a KB test

• recommend 2 vials of RhIG

• suggest that RhIG is not necessary because records indicate that the mother received prenatal RhIG

• recommended one vial of RhIG because it is below the cutoff for the fetal screen

submit the sample for a KB test

When a mother’s plasma contains a clinically significant antibody, what test must be done to determine its concentration?

an antibody titer