11.4 Hemolytic Disease of the Fetus and Newborn (HDFN) and Rh Immune Globulin

Hemolytic Disease of the Fetus and Newborn (HDFN) and Rh Immune Globulin

Hemolytic disease of the fetus and newborn links three blood bank skills that the board tests together: recognizing which maternal antibodies are dangerous, monitoring an alloimmunized pregnancy, and preventing anti-D formation with correctly dosed Rh immune globulin (RhIg). It also covers the neonatal end of the problem: cord blood testing and choosing blood for intrauterine and exchange transfusion. Rh(D)-negative mothers are uncommon among Filipinos, so ABO HDFN is the form most often seen in Philippine nurseries, but anti-D prophylaxis and fetomaternal hemorrhage calculations remain standard board material.

---

1. Pathophysiology and Rh Versus ABO HDFN

Pathophysiology of Maternal Alloimmunization

HDFN develops when maternal IgG antibodies directed against a paternal erythrocyte antigen inherited by the fetus cross the placenta via the neonatal Fc receptor (FcRn). The IgG antibodies bind fetal red blood cells, which are subsequently destroyed via extravascular clearance by fetal splenic macrophages.

┌────────────────────────────────────────────────────────────────────────────────────────┐
│                     Pathophysiological Cascade of In Utero HDFN                        │
├────────────────────────────────────────────────────────────────────────────────────────┤
│ Maternal IgG Alloantibody (e.g., anti-D) crosses placenta via FcRn                     │
│                                       │                                                │
│                                       ▼                                                │
│ Binds fetal antigen-positive red blood cells → Splenic macrophage clearance        │
│                                       │                                                │
│                                       ▼                                                │
│ Severe Fetal Anemia → Extramedullary hematopoiesis (Hepatosplenomegaly)             │
│                                       │                                                │
│                                       ▼                                                │
│ Hepatic portal hypertension + Hypoalbuminemia + High-output cardiac failure            │
│                                       │                                                │
│                                       ▼                                                │
│ HYDROPS FETALIS (Generalized anasarca, pericardial effusion, ascites, fetal demise)    │
├────────────────────────────────────────────────────────────────────────────────────────┤
│                             Post-Delivery Neonatal Cascade                             │
│                                                                                        │
│ In utero: Maternal placenta clears unconjugated bilirubin.                             │
│ At birth: Massive ongoing hemolysis + Immature neonatal UDP-glucuronosyltransferase.   │
│ Rapid surge in unconjugated bilirubin → Crosses immature blood-brain barrier       │
│                                       │                                                │
│                                       ▼                                                │
│ KERNICTERUS (Bilirubin encephalopathy, basal ganglia necrosis, permanent brain damage) │
└────────────────────────────────────────────────────────────────────────────────────────┘

Comparative Analysis: Rh HDFN vs. ABO HDFN

---

2. Rh Immune Globulin (RhIg) Prophylaxis and Fetomaternal Hemorrhage Calculations

Rh Immune Globulin (RhIg or RhoGAM) is a sterile solution containing high titers of human anti-D IgG. Administering exogenous anti-D to an unsensitized Rh-negative mother masks foreign fetal Rh-positive red cells and induces rapid immune clearance through antibody-mediated immune suppression, preventing active maternal B-cell alloimmunization.

Regulatory Dosing and Indications

• Standard Dose: $300\ \mu\text{g}$ ($1,500\text{ IU}$) of anti-D.
• Coverage: One standard $300\ \mu\text{g}$ vial neutralizes up to $30\text{ mL}$ of fetal Rh-positive whole blood (equivalent to $15\text{ mL}$ of packed red blood cells).
• Microdose: A $50\ \mu\text{g}$ dose is indicated for pregnancy termination, ectopic pregnancy, or chorionic villus sampling occurring prior to $12\text{ weeks}$ of gestation (fetal blood volume is $<5\text{ mL}$).
• Routine Prophylaxis Schedule:
  1. Antenatal: Administered at $28\text{ weeks}$ gestation to all unsensitized Rh-negative pregnant females.
  2. Postpartum: Administered within $72\text{ hours}$ of delivery if the newborn is confirmed Rh (D)-positive (or weak D-positive) and the maternal antibody screen confirms absence of active anti-D alloimmunization.

Fetomaternal Hemorrhage (FMH) Assessment

Normal delivery involves an average fetomaternal bleed of $<5\text{ mL}$. However, obstetrical trauma, placental abruption, or manual extraction can trigger massive fetomaternal hemorrhages exceeding $30\text{ mL}$, necessitating quantitative evaluation and multiple RhIg vials.

1. Qualitative Screening: The Rosette Test:
   • Maternal postpartum blood is incubated with reagent anti-D, washed, and mixed with enzyme-treated Rh-positive indicator red blood cells.
   • Rosettes (clusters of indicator cells surrounding antibody-coated fetal cells) form. The test reliably detects fetomaternal hemorrhage of $\ge 10\text{ mL}$.
   • If negative, 1 standard $300\ \mu\text{g}$ vial is administered.
   • If positive, quantitative testing is mandatory to calculate the exact number of vials required.
2. Quantitative Determination: The Kleihauer-Betke (KB) Acid Elution Test:
   • Biochemical Principle: A thin maternal blood smear is fixed with ethanol and immersed in an acid elution buffer ($\text{pH } 3.2\text{ to }3.3$). Adult maternal hemoglobin (HbA) is soluble in acid and elutes out of the cells, leaving behind pale, translucent erythrocyte membranes ("ghost cells"). Fetal hemoglobin (HbF) is resistant to acid elution and remains trapped within the cell. Counterstaining with eosin or erythrosin stains fetal red blood cells dark pink/red.

                              ┌────────────────────────────────────────┐
                              │   Kleihauer-Betke Acid Elution Smear   │
                              │         (Count 2,000 Total Cells)      │
                              └───────────────────┬────────────────────┘
                                                  │
                                                  ▼
                              ┌────────────────────────────────────────┐
                              │      Step 1: % Fetal Cells             │
                              │      % = (Fetal Cells / 2,000) x 100   │
                              └───────────────────┬────────────────────┘
                                                  │
                                                  ▼
                              ┌────────────────────────────────────────┐
                              │   Step 2: Fetal Whole Blood (mL)       │
                              │   Volume = % Fetal Cells x 50          │
                              └───────────────────┬────────────────────┘
                                                  │
                                                  ▼
                              ┌────────────────────────────────────────┐
                              │    Step 3: Calculate RhIg Vials        │
                              │    Vials = (Fetal Blood Volume / 30)   │
                              └───────────────────┬────────────────────┘
                                                  │
                                                  ▼
                              ┌────────────────────────────────────────┐
                              │       Step 4: Standard Rounding        │
                              │   • Decimal <0.5: Round DOWN + 1 vial  │
                              │   • Decimal ≥0.5: Round UP + 1 vial    │
                              └────────────────────────────────────────┘

The Kleihauer-Betke Mathematical Formulas and Rounding Rules

$\%\text{ Fetal Cells} = \left( \frac{\text{Number of Fetal Cells Counted}}{2,000\text{ Total Maternal Cells}} \right) \times 100$

$\text{Volume of Fetal Whole Blood (mL)} = \%\text{ Fetal Cells} \times 50$

$\text{Calculated Number of } 300\ \mu\text{g RhIg Vials} = \frac{\text{Volume of Fetal Whole Blood (mL)}}{30\text{ mL}}$

Strict Blood Banking Rounding Rules for RhIg Dosing: To prevent catastrophic maternal alloimmunization from under-dosing, analyze the decimal value resulting from dividing by 30:

• If the number to the right of the decimal point is less than $0.5$ ($<0.5$), round down to the nearest whole integer, and add $1$ safety vial.
• If the number to the right of the decimal point is greater than or equal to $0.5$ ($\ge 0.5$), round up to the next whole integer, and add $1$ safety vial.

• Worked Example A ($<0.5$ decimal):
  • Fetal cells counted: $16$ out of $2,000$.
  • $\%\text{ Fetal Cells} = (16 / 2,000) \times 100 = 0.8\%$.
  • $\text{Fetal Whole Blood} = 0.8 \times 50 = 40\text{ mL}$.
  • $\text{Vials} = 40 / 30 = 1.33$.
  • Because $.33 < 0.5$, round down to $1$, then add $1$ safety vial: $1 + 1 = \mathbf{2\text{ vials}}$ ($600\ \mu\text{g}$).
• Worked Example B ($\ge 0.5$ decimal):
  • Fetal cells counted: $35$ out of $2,000$.
  • $\%\text{ Fetal Cells} = (35 / 2,000) \times 100 = 1.75\%$.
  • $\text{Fetal Whole Blood} = 1.75 \times 50 = 87.5\text{ mL}$.
  • $\text{Vials} = 87.5 / 30 = 2.92$.
  • Because $.92 \ge 0.5$, round up to $3$, then add $1$ safety vial: $3 + 1 = \mathbf{4\text{ vials}}$ ($1,200\ \mu\text{g}$).

---

3. Prenatal Monitoring of an Alloimmunized Pregnancy

1. First prenatal visit: ABO and Rh(D) typing and an antibody screen for every pregnant patient, whatever her D type.
2. Positive screen: Identify the antibody and decide whether it can cause HDFN. Clinically significant antibodies are IgG and react with antigens already present on fetal red cells. Anti-D, anti-c, and anti-K cause the most severe disease. Anti-Lea, anti-Leb, anti-I, and anti-P1 are usually IgM, and Lewis antigens are poorly developed at birth, so they do not cause HDFN.
3. Antibody titration: The titer is measured with an antiglobulin tube method, and the current sample is tested in parallel with the previous frozen sample so a true rise is not confused with run-to-run variation. Each laboratory defines its critical titer, commonly 16 to 32 for anti-D. A titer at or above the critical value triggers fetal assessment.
4. Fetal assessment: Middle cerebral artery peak systolic velocity (MCA-PSV) Doppler has largely replaced serial amniocentesis and the Liley ΔA450 curve for detecting fetal anemia. Fetal RHD genotyping from cell-free fetal DNA in maternal plasma, or paternal antigen typing, tells whether the fetus is at risk at all.
5. Anti-K warning: Anti-K suppresses fetal erythropoiesis as well as destroying red cells, so the titer and the bilirubin level can underestimate how anemic the fetus is.

---

4. Cord Blood Testing and Blood Selection for the Newborn

Cord Blood Workup

• ABO forward typing only. Newborns have not yet made their own anti-A or anti-B, and any antibodies present are maternal IgG, so reverse typing is not performed.
• Rh(D) typing, including a weak D test when it decides RhIg eligibility for a D-negative mother.
• Direct antiglobulin test (DAT). A strongly positive DAT supports Rh HDFN. In ABO HDFN the DAT is often weak or negative because fetal red cells carry few A and B sites. An eluate tested against A1 and B cells shows which maternal antibody is coating the cells.
• Pitfall: Wharton's jelly from the umbilical cord can cause false agglutination. Wash cord cells thoroughly before testing.

Blood for Intrauterine and Exchange Transfusion

• Group O red cells (or cells compatible with both mother and infant) that lack the antigen targeted by the maternal antibody, usually D-negative in Rh HDFN.
• Crossmatch-compatible with the mother's plasma, because the antibody causing the disease is maternal.
• Irradiated to prevent transfusion-associated graft-versus-host disease, CMV-safe (leukoreduced or CMV-seronegative), and hemoglobin S-negative.
• Fresh red cells (commonly less than 5 to 7 days old) to limit the potassium load. For exchange transfusion, red cells are often reconstituted with group AB plasma.
• A double-volume exchange transfusion removes most antibody-coated red cells (about 85%) and lowers the bilirubin by roughly half, preventing kernicterus. Phototherapy converts unconjugated bilirubin to water-soluble isomers that are excreted without conjugation.