Alloimmunization Part II: Management

What to do with a positive antibody screen?

• First, obtain a antibody titer: a value of dilution at which the antibody screen remains positive.

  • So a 1:32 titer is more significant than a 1:4 titer.

  • A critical titer is one that carries risk for hydrops, which is generally between 1:8 - 1:32, depending on the lab.

    • Additionally, a change in titer of more than one dilution is considered significant. 

  • For titers less than 1:8, titers should be obtained every 4 weeks to reassess.

    • Exception: anti-Kell antibodies, as these titers do not correlate with fetal status. 

• Assess paternal genotype.

  • If the infant can’t get the antigen that the mother is sensitized to (i.e., dad has no Kell antigen for mom’s anti-Kell antibodies to attack), then there’s no need for assessment.

  • If the father does carry an offending antigen, DNA testing can be utilized to determine if the father is heterozygous or homozygous. 

• Assess fetal DNA.

  • Can be employed when paternal status is unknown, or carries risk.

  • Amniocentesis is the gold-standard methodology for fetal blood typing; CVS can also be employed, but carries a higher risk of de novo alloimmunization than amniocentesis.

  • Rh (D) antigen carriage can also be determined by cell free fetal DNA assays.

Monitoring the Alloimmunized Pregnancy

• Historically: serial amniocentesis

  • The concentration of bilirubin in amniotic fluid is assessed by spectral analysis through 450 nm light (OD450)

• Currently: middle cerebral artery (MCA) Doppler ultrasonography

  • Studies have correlated the relationship between peak systolic velocity 1.5 times the median for gestational age with moderate-to-severe fetal anemia, with 100% sensitivity.

    • However, this monitoring should be done by those with experience in the technique, as even with good technique the false positive rate approaches 12%.

• For minor blood group antigen sensitization, protocols may be different.

  • Anti-Kell in particular has a less predictable course and often results in more severe fetal anemia than alloimmunization due to other blood antigens. 

• If severe anemia is suspected, periumbilical cord blood sampling (PUBS, aka cordocentesis) can be used to measure the fetal hemoglobin directly.

  • An intrauterine transfusion (IUT) can be performed to transfuse the fetus as well. 

Interventions and delivery

• Delivery timing is controversial:

  • ACOG recommends if only mild hemolysis is suspected, induction can be considered at 37-38 weeks.

  • With more severely sensitized pregnancies, the risk of repeated cord blood sampling and intrauterine transfusion has to be compared to that of early delivery, and this is often dependent on the center and their experience. 

Alloimmunization Part I: Basics, Prevention

Further Reading
PB 181 - Preventing Rh Alloimmunization
PB 192 - Management of Alloimmunization

The basics

• Over 36 blood group systems for human blood, but the most commonly used are:

  • the ABO system

  • the Rhesus (or Rh) blood group system. 

• The Rh system has over 40 blood group antigens; of these, the most important are “D,” “C,” “c,” “E,” and “e.” (There is no “d.”)

  • The D group is what we most commonly think about.

  • Someone who is Rh+ refers to having a positive Rh(D) antigen. 

  • Antibodies to Rh(D) and Rh(c) group antigens confer significant risk of hemolytic disease of the newborn.

• Rh negative status is more common amongst white persons (15%).

  • In contrast, 5-8% of black persons are Rh(D) negative.

  • 1-2% of Asians and Native Americans are Rh(D) negative.

  • In white Rh(D) positive individuals, 60% are heterozygous and 40% are homozygous at the D locus. 

• Beyond the Rh system blood antigens, there are also a number of other significant or common blood group antigens encountered in clinical practice, such as:

  • “Lewis” (‘Lea’ and ‘Leb’) antigens

  • “I” antigens

  • “Kell” antigens.

  • PB 192 has a big table of these atypical antibodies and their relationship to fetal hemolytic disease.

Alloimmunization

• Alloimmunization or isoimmunization (synonymous) is the formation of maternal antibodies against blood group antigens not possessed by the mother

  • For instance, an Rh-negative mother developing anti-Rh(D) antibodies if exposed to Rh-positive blood.

  • This can occur if a sufficient number of erythrocytes from an Rh-positive fetus gain access to the circulation of an Rh-negative mother. 

• Hemorrhage sufficient enough to cause alloimmunization most commonly occurs at delivery (15-50%)

• But only a small amount of bleeding across this interface (as little as 0.1 mL!) is sufficient to cause alloimmunization.

  • Only 1-2% Rh alloimmunization occurs as a consequence of antepartum bleeding or trauma.

  • Alloimmunization has been reported after ectopic pregnancy, threatened abortion, and spontaneous or induced abortion as well.

  • Obstetrical procedures may also predispose to fetomaternal hemorrhage, such as CVS (14% risk), amniocentesis (2-6%), cordocentesis/PUBS, or external cephalic version (2-6%). 

• Rh alloimmunization was formerly a much more common pregnancy complication that has considerably decreased in incidence in the US and other countries, from 13-16% of pregnancies pre-1970s, to 0.14-0.2% today.

  • Pregnancy complication that in the US is almost completely avoidable with good prenatal care and adherence to protocols. 

• Rh alloimmunization predisposes to Rh hemolytic disease of the newborn: 

• Generally, a sensitizing pregnancy (1st event) is unaffected as antibodies are created in maternal serum from the first exposure to Rh(+) antigen. 

• Subsequent infants with Rh(+) types will be affected by disease as antibodies in maternal serum cross and attack fetal red cells. 

• This in turn puts the fetal hematopoietic system into overdrive, causing organomegaly of the liver and spleen (due to shifting production of immature red cells to these organs). 

• This will ultimately cause portal hypertension and failure of the liver to make other products such as albumin, which then will cause a high output cardiac failure and hydrops. 

• In areas where prophylaxis and treatments are not standard/available, 14% of affected fetuses are stillborn, and up to 50% of live-born infants suffer neonatal death or brain injury from kernicterus (hyperbilirubinemia) as a result of this disease.

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Wikipedia.

Prevention of Rh(D) Alloimmunization

• Women should be tested for ABO blood group and RhD type and screened for presence of any erythrocyte antibodies at entry to care.

  • This should be repeated with each subsequent pregnancy. 

• Assuming a negative antibody screen for anti-D antibodies, Rh negative patients should be considered for anti-D immune globulin, better known as Rhogam, at 28 weeks gestation.

  • Since the 1970s, the strategy in the United States has been to prevent RhD alloimmunization by universal administration of anti-D immune globulin. 

    • Studies looking at cost-effectiveness of selective administration based on the partner’s blood type versus universal administration have shown the approaches to be cost equivalent.

    • Cell free fetal DNA testing is another approach that is actively being studied in the literature and considered to help reduce exposure to RhIg, which is a human blood product; its sensitivity is 99% and specificity 95%, though this is variable to some degree with respect to race. 

• Postpartum administration of RhIg brings the risk of alloimmunization from 16% (with no RhIg) to just over 2%.

  • RCTs that examined RhIg versus placebo administration postpartum had an astonishingly high risk reduction of 88-96%, depending on the time period after the pregnancy studied. 

• The single 300 mcg dose of RhIg at 28 weeks gestation reduces the risk of new 3rd trimester alloimmunization even further:

  • Since the introduction of universal RhIg at 28 weeks in addition to postpartum RhIg, Rh alloimmunization rates went from over 2% to less than 0.2%. 

• RhIg should also be given within 72 hours of a sensitizing event -- that is, one that causes or potentially causes bleeding at the fetal-maternal interface.

  • 1st trimester or early 2nd trimester event such as a procedure, an ectopic, a threatened or complete miscarriage or abortion, the overall risk of alloimmunization is low.

    • Fetal red cell volume is only about 1.5 cc at 12 weeks (3cc total blood volume).

    • 50 - 120 mcg RhIg before 12 weeks.

    • 300mcg dose for events after 12 weeks. 

  • Late 2nd or 3rd trimester bleeding events have a more significant risk for alloimmunization, as fetal blood volume is higher.

    • In this case, a type-and-screen should be obtained, as well as a rosette and a Kleihauer-Betke test.

      • Rosette qualitative screen that can detect greater than 2mL of fetal whole blood in circulation.

        • By incubating a maternal blood sample with Rh immunoglobulin, this will bind any present Rh-positive fetal cells, forming “rosettes” that can then be seen on microscopy.

        • If a rosette test is positive, the next step is to perform a quantitative assessment such as the KB test or flow cytometry.

      • KB test measures the amount of fetal hemoglobin in the mother’s circulation, and thus estimates the amount of RhIg needed to prevent alloimmunization.

        • Results are reported as the number of fetal erythrocytes in maternal blood per 2,000 red blood cells. This often is reported as a percentage of fetal red cells in maternal blood volume (i.e., 200/2000 = 0.1, or 10%).

        • Because KB tests utilize a stain looking for fetal hemoglobin, they are less accurate in hemoglobinopathies that increase fetal hemoglobin in maternal red cells (such as sickle-cell disease or thalassemias).

      • Flow cytometry uses monoclonal antibodies to detect hemoglobin F or RhD antigens, and is also very sensitive and accurate in detecting fetal cells in maternal blood. 

    • 300 mcg of RhIg (or one vial) is sufficient enough to cover against up to 30 mL of fetal blood; up to 8 vials can be administered every 12 hours until reaching the desired dosage -- and fortunately, there’s an IV form if you need larger quantities! 

• Other important facts to know about Rhogam:

  • RhIg has a half life of about 23 days, and thus a 300mcg dose is detectable for approximately 12 weeks. 

  • Redosing may be held if delivery or subsequent sensitizing events occur within 3 weeks (or 1 half life) of a dose; redosing should be more strongly considered after this interval. 

  • After delivery, given the window of 72 hours for effective administration, it’s reasonable to hold off on re-dosing RhIg until Rh type results are available on the infant; if it is Rh negative, there is no indication for additional dosing.

  • RhIg is a human blood product, collected from volunteer donors who have high titers of anti-Rh(D) antibodies. There are no available synthetic/recombinant forms or alternative medications currently available. 

  • Rhogam only prevents Rh(D) alloimmunization by neutralizing the antigen; it doesn’t work on those who have already been sensitized to Rh(D) antigen.

One classic CREOG question involves calculating the amount of RhIg needed to prevent alloimmunization from a KB test. 

• Often, these questions supply the volume of fetal bleed, rather than a KB, which makes life a lot easier.

• Since 300 mcg covers up to 30 cc fetal blood, if the fetal bleeding volume is known, you just need to set up a proportion:

  • 30 mL fetal blood / 300 mcg RhIg = __ mL fetal blood (in question) / x mcg RhIg.

• If the volume of fetal bleeding is not supplied and you only have the KB result, you must first calculate the fetal bleeding volume based on maternal weight. The maternal blood volume is expected to be 70 cc/kg.

  • Maternal blood volume = 70 mL / kg x __ kg (maternal weight).

  • KB % x maternal blood volume = fetal bleed volume detected.