4.5 Hemolytic Disease of the Fetus and Newborn (HDFN) & RhoGAM

Hemolytic Disease of the Fetus and Newborn (HDFN)

HDFN is a pathological condition where the lifespan of fetal or neonatal red blood cells is shortened due to the binding of maternal alloantibodies. For HDFN to occur, three strict biological conditions must be met:

1. Maternal Antibody: The mother must have a pre-existing IgG antibody, or develop one during the pregnancy. Only the IgG class of immunoglobulins can be actively transported across the placenta by Fc receptors.
2. Fetal Antigen: The fetus must possess the specific red cell antigen corresponding to the maternal antibody. This antigen is inherited from the father.
3. Antigen Expression: The antigen must be well-developed on the fetal red blood cells during gestation so the antibodies can bind.

Pathophysiology of HDFN

When maternal IgG coats the fetal red cells, the cells are not usually destroyed intravascularly. Instead, they are destroyed by macrophages in the fetal spleen (extravascular hemolysis). This continuous destruction leads to profound fetal anemia.

In Utero (The Fetal Stage): To compensate for the anemia, the fetal liver and spleen vastly increase red cell production (extramedullary hematopoiesis), leading to massive hepatosplenomegaly. As the liver focuses on making red cells, it decreases protein synthesis, leading to hypoproteinemia. This causes a decrease in oncotic pressure, resulting in widespread edema, ascites, and eventual heart failure in the fetus, a lethal condition known as hydrops fetalis.

Post-Partum (The Neonatal Stage): Before birth, the mother's liver processes the bilirubin generated by the fetal hemolysis. After birth, the newborn's immature liver lacks the enzyme glucuronyl transferase required to effectively conjugate the massive amounts of excess indirect bilirubin. Unconjugated bilirubin is lipid-soluble and highly toxic; it crosses the blood-brain barrier and deposits in the basal ganglia, leading to severe neurological damage or death, a condition known as kernicterus. Treatment involves intense phototherapy (UV light alters the bilirubin structure to be water-soluble for excretion) or an exchange transfusion to remove bilirubin and maternal antibodies while providing compatible red cells.

Types of HDFN

There are two primary categories of HDFN, differing greatly in presentation:

ABO HDFN

• Most Common Setup: Group O mother, Group A or B infant.
• Frequency: The most common cause of HDFN.
• Severity: Usually mild. The A and B antigens are not fully developed on fetal red cells at birth. Furthermore, A and B antigens are widely distributed on other tissue cells throughout the body, which act as a "sink," absorbing the maternal antibody before it can reach the red blood cells.
• Occurrence: Can occur in the first pregnancy without prior sensitization because group O individuals naturally produce high-titer IgG anti-A,B without prior exposure to foreign blood.
• DAT: Weakly positive or negative. Elution may be required to prove the antibody's presence on the cells.

Rh HDFN (Anti-D)

• Setup: Rh-negative mother, Rh-positive infant.
• Severity: Can be extremely severe, leading to hydrops fetalis or death.
• Occurrence: Rarely occurs in the first pregnancy. The mother must first be immunized (sensitized) to the D antigen, typically via a fetal-maternal hemorrhage during the delivery of her first Rh-positive baby. Subsequent Rh-positive pregnancies are at extreme risk as the mother's immune system mounts a massive anamnestic IgG response.
• DAT: Strongly positive.
• Note: Other IgG antibodies (like anti-K, anti-c, anti-Fya) can also cause severe HDFN. Anti-Kell (anti-K) is particularly severe because it targets erythroid precursors in the bone marrow, suppressing red cell production in addition to causing hemolysis.

Fetal Interventions and Cord Blood Testing

If severe HDFN is detected via Middle Cerebral Artery (MCA) Doppler ultrasound or amniocentesis (plotted on a Liley graph), an intrauterine transfusion (IUT) may be required. Blood provided for an IUT must be highly specialized: Group O, Rh negative, CMV negative, Leukoreduced, Irradiated, Hemoglobin S negative, and fresh (<7 days old) to maximize 2,3-DPG.

At birth, cord blood from infants born to O positive or Rh negative mothers is tested. A DAT is performed. If the cord blood requires ABO typing, it must be washed thoroughly to remove Wharton's jelly, a hyaluronic acid-rich substance that causes false-positive spontaneous agglutination.

Rh Immune Globulin (RhIG / RhoGAM)

RhIG is a concentrated, sterile formulation of human anti-D IgG. Its administration to an Rh-negative mother is a form of passive immunity. The injected anti-D attaches to any fetal Rh-positive red cells that entered the maternal circulation, clearing them in the maternal spleen before the mother's immune system can recognize the D antigen and form her own active, permanent anti-D.

Indications for RhIG: An Rh-negative mother is a candidate for RhIG if:

1. She is pregnant or has just delivered a baby.
2. The baby is Rh-positive (or the baby's type is unknown, such as after a miscarriage).
3. The mother has not already formed active anti-D (she is not sensitized). If the antibody screen is positive for anti-D, an investigation must determine if it is residual RhIG from a prophylactic antenatal dose or true active immunization.

Standard Administration Schedule:

• Antenatal: 1 dose at 28 weeks gestation.
• Postpartum: 1 dose within 72 hours of delivery of an Rh-positive infant.

Detecting and Quantifying Fetal-Maternal Hemorrhage (FMH)

At delivery, a maternal blood sample is drawn to determine if the standard single dose of RhIG is sufficient, or if a massive hemorrhage requires multiple vials.

1. Rosette Test (Fetal Screen): A qualitative screening test. Maternal blood is incubated with anti-D, which binds to any fetal Rh-positive cells present. D-positive indicator cells are then added, which bind to the anti-D coating the fetal cells, forming visible "rosettes" around the central fetal cell under a microscope. A positive test indicates a significant FMH (>30mL) has occurred, mandating a quantitative test.

2. Kleihauer-Betke (KB) Test: A quantitative test based on the principle that fetal hemoglobin (HbF) is highly resistant to acid elution, while adult hemoglobin (HbA) is readily eluted. A maternal blood smear is treated with an acid bath and stained.

   • Fetal red cells (containing HbF) retain the stain and appear bright pink.
   • Adult maternal red cells (containing HbA) lose their hemoglobin and appear as pale "ghosts." By counting 2000 cells to find the percentage of fetal cells, the exact volume of the bleed is calculated.

The Kleihauer-Betke Calculation Rules: One standard 300 µg dose of RhIG protects against a fetal bleed of 30 mL of whole blood (or 15 mL of packed red cells).

Step 1: Calculate the volume of fetal whole blood bleed. Percentage of fetal cells × 50 = Volume of fetal hemorrhage in mL. (The 50 factor represents an assumed maternal blood volume of 5000 mL divided by 100%).

Step 2: Calculate the base number of vials needed. Volume of fetal hemorrhage / 30 mL = Number of vials.

Step 3: Apply the strict AABB rounding rule.

• If the decimal is < 0.5, round down to the whole number, and add 1 safety vial.
• If the decimal is ≥ 0.5, round up to the next whole number, and add 1 safety vial. (Note: Always adding the safety vial ensures adequate coverage for the margin of error in the manual KB count).

Example calculation: A KB test shows 1.8% fetal cells.

1. 1.8 × 50 = 90 mL fetal bleed.
2. 90 / 30 = 3.0 vials.
3. Because the decimal is <0.5 (it is exactly .0), round down to 3, and add 1 safety vial. Total = 4 vials required.