M9 - HDFN and AIHA

Define HDFN

HDFN is the destruction of the red blood cells of the foetal or neonatal RBCS by antibodies produced by the mother

Which maternal antibody class is capable of crossing the placenta and causing HDFN

IgG

What was the main cause of HDFN prior to 1968?

Anti-D against the Rh antigen

How did the incidence of HDFN caused by anti-D antibodies change after 1968, and why?

It decreased due to the introduction of the RH immunoglobulin prophylaxis

Explain why only certain foetal RBC antigens trigger HDFN.

The foetal RBC antigens that can trigger HDFN are those inherited from the father that are foreign to the mother’s immune system.

Which type of RBC incompatibility is currently more frequently observed than anti-D in referral centres?

Other RBC antigen incompatibilities (Non-D antigens)

Describe the mechanism which maternal antibodies cause HDFN

Maternal antibodies, mainly the IgG subclass, cross the placenta, binds to the foreign foetal RBC antigens, and mediate their destruction by the reticuloendothelial system

Why is Rh(D) incompatibility common, yet HDFN incidence has decreased?

Use of RhIG prevents maternal sensitization and HDFN

Differentiate between the maternal and foetal roles in HDFN.

The mother produces IgG antibodies against paternal RBC antigens; the fetus expresses these antigens on RBCs, which are then targeted for destruction.

Explain why the first pregnancy with a D-positive fetus rarely results in severe HDFN.

The mother usually becomes sensitized during the first pregnancy, so significant antibody production affecting the fetus is more likely in subsequent pregnancies.

List two clinical implications of maternal-foetal RBC incompatibility.

• Fetal anemia

• Neonatal jaundice or hyperbilirubinemia

Why is the firstborn Rh-positive infant of an Rh-negative mother usually unaffected by HDFN?

Because the mother has not yet been immunized to the D antigen.

How are maternal anti-D antibodies produced?

Fetal Rh-positive RBCs enter the maternal circulation during gestation or delivery, stimulating maternal immune response and production of anti-D antibodies.

How does maternal immunization affect subsequent pregnancies?

All subsequent Rh-positive fetuses are at risk of HDFN because the mother already has anti-D antibodies.

How does ABO incompatibility between mother and fetus influence Rh immunization?

Major ABO incompatibility reduces the incidence of detectable feto-maternal hemorrhage and can protect against Rh immunization.

What is the approximate incidence of transplacental haemorrhage during gestation and at delivery?

Up to 7% during gestation; more than 50% at delivery.

Name three interventions or events that can increase the risk of feto-maternal hemorrhage.

Amniocentesis, chorionic villus sampling, abdominal trauma.

Which IgG subclasses are most efficient in causing RBC hemolysis in HDFN?

IgG1 and IgG3.

Which non–Rh RBC antibody is most clinically significant in causing HDFN?

Anti-Kell.

Describe the mechanism of foetal RBC destruction in Rh HDFN.

Maternal IgG binds to specific antigens on foetal RBCs, leading to their destruction by the foetal reticuloendothelial system.

What factors influence the rate of fetal RBC destruction?

• Antibody titer, antibody specificity, and number of RBC antigenic sites.

What is “erythroblastosis fetalis”, and why does it occur?

The presence of immature RBCs (erythroblasts) in fetal circulation due to accelerated RBC production by the fetal bone marrow in response to anemia.

What causes hepatosplenomegaly in HDFN?

Extramedullary hematopoiesis in the liver and spleen due to increased RBC production

Explain the pathophysiology of hydrops fetalis.

Severe anemia and hypoproteinemia lead to high-output cardiac failure, generalized edema, effusions, and ascites.

Why does the rate of RBC destruction decrease after birth in affected neonates?

No additional maternal antibodies enter the neonate’s circulation; destruction continues only as maternal IgG gradually decays based on its half-life.

How is bilirubin produced in HDFN?

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

How does maternal metabolism affect foetal bilirubin levels?

Maternal liver metabolises unconjugated bilirubin, protecting the foetus in utero.

Why are newborns at risk of hyperbilirubinemia after birth?

The immature neonatal liver cannot adequately conjugate unconjugated bilirubin.

At what bilirubin levels does the risk of neurotoxicity (kernicterus) increase?

Generally when unconjugated bilirubin exceeds 18–20 mg/dL.

What is kernicterus?

Brain dysfunction caused by toxic levels of unconjugated bilirubin.

When is the recommended time to perform type & antibody screening in pregnancy?

At the first prenatal visit, preferably in the first trimester.

What maternal history is important for HDFN risk assessment?

Previous pregnancies, their outcomes, and prior transfusions.

What laboratory parameters are included in prenatal testing for HDFN?

ABO group, Rh type, and antibody screening.

What characteristics must the antibody screen detect?

Clinically significant IgG alloantibodies reactive at 37°C and in the antiglobulin phase.

What is the next step if the antibody screen is reactive?

Determine the specific antibody identity.

How does antibody specificity affect follow-up testing?

Testing depends on the antibody’s specificity and likely clinical significance.

How is anti-D prophylaxis administered in Rh-negative pregnant women?

Either after events increasing feto-maternal hemorrhage risk or antenatally at 28 and 34 weeks’ gestation.

What is the purpose of antibody titration in pregnancy?

To determine the relative concentration of antibodies capable of crossing the placenta and causing HDFN.

Which testing phase is required for accurate titration?

• The indirect antiglobulin (Coombs) phase using anti-IgG reagent.

How is the titer expressed?

As the reciprocal of the titration endpoint or as a titer score.

What titer value indicates a need to assess fetal anemia?

A titer reproducibly at 1:32 or above.

What noninvasive method is used to predict fetal anemia?

Measurement of middle cerebral artery peak systolic velocity (MCA-PSV) using Doppler ultrasonography.

How does MCA-PSV relate to fetal anemia?

Increased blood velocity in the middle cerebral artery correlates with anemia.

When might earlier interventions be required in HDFN pregnancies?

In patients with a history of a severely affected fetus or early fetal death.

Why is amniocentesis now rarely used in HDFN management?

MCA-PSV is noninvasive and provides equivalent information on fetal anemia.

How can amniotic fluid bilirubin measurement predict fetal HDFN severity?

By measuring bilirubin concentration (∆OD 450 nm) as pregnancy progresses, indicating worsening hemolysis.

What are the main indications for intrauterine transfusion in HDFN?

• MCA-PSV indicating fetal anemia

• Ultrasound evidence of fetal hydrops

• Fetal hemoglobin <100 g/L

• High amniotic fluid ∆OD 450 nm results

Why must the risks and benefits of intrauterine transfusion be carefully weighed?

Because IUT carries risks such as fetal bradycardia, preterm labor, infection, or fetal death, but can prevent severe anemia and hydrops fetalis.

What type of RBCs are typically selected for intrauterine transfusion?

Group O RBCs that are antigen-negative for maternal antibodies, CMV-negative, and ideally <7 days old.

Explain why RBCs used in IUT must be antigen-negative for maternal antibodies.

To prevent further hemolysis of transfused cells by maternal antibodies.

What is the mechanism of phototherapy in neonatal hyperbilirubinemia?

Light at 460–490 nm converts unconjugated bilirubin into water-soluble isomers that are less lipophilic and less neurotoxic.

How does IVIG reduce hemolysis in neonates with HDFN?

IVIG competes with maternal IgG for Fc receptors on splenic macrophages, reducing RBC destruction.

What are the primary and secondary purposes of exchange transfusion in HDFN?

• Primary: Remove high levels of unconjugated bilirubin to prevent kernicterus

• Secondary: Remove maternal antibodies, sensitized RBCs, and replace with compatible RBCs

Which serologic tests are used on cord blood to confirm HDFN?

ABO grouping, Rh typing, Direct Antiglobulin Test (DAT), and elution.

What is the purpose of RhIG in pregnancy?

To prevent maternal active immunization against D antigen and formation of anti-D antibodies.

What is the approximate risk of sensitization in an Rh-negative mother carrying an Rh-positive fetus without prophylaxis?

Up to 16%.

When should postpartum RhIG be administered?

Within 72 hours of delivery of an Rh-positive infant to a non-immunized Rh-negative mother.

How does the Kleihauer-Betke test guide postpartum RhIG dosing?

It estimates the volume of fetal hemorrhage into maternal circulation to calculate the required dose.

Why is RhIG ineffective in a mother who has already been actively immunized?

Because she has already produced anti-D antibodies; RhIG cannot reverse active immunization.

Which infants are typically affected by ABO HDFN?

Group A or B infants born to group O mothers.

How does ABO HDFN differ from Rh HDFN regarding pregnancy order?

ABO HDFN can occur in the first pregnancy, whereas Rh HDFN usually affects subsequent pregnancies after maternal sensitization.

Why is there no single serologic test diagnostic for ABO HDFN?

Because the disease is usually mild, and antibody titers can be low or variable; multiple tests including DAT, maternal and cord blood typing are needed.

What is the recommended practice for detecting ABO HDFN postnatally?

Collect cord blood samples on all delivered infants and perform serologic testing.

A fetus shows MCA-PSV consistent with severe anemia, but amniotic fluid ∆OD 450 nm is normal. What would be the clinical implication?

MCA-PSV is a more direct and noninvasive measure of fetal anemia; IUT may still be indicated despite normal ∆OD 450 nm.

After birth, a neonate with HDFN has a bilirubin of 22 mg/dL. Which management options are indicated?

Phototherapy, IVIG to reduce haemolysis, and possibly exchange transfusion if bilirubin continues to rise.

Explain how maternal anti-D prophylaxis indirectly reduces the need for intrauterine transfusions.

By preventing maternal sensitisation, subsequent foetuses are protected from HDFN, reducing the incidence of severe foetal anaemia requiring IUT.

Define immune haemolytic anaemia and list its three broad categories.

• Definition: Shortened red blood cell (RBC) survival mediated through the immune response, specifically by humoral antibodies.

• Categories:

  1. Alloimmune

  2. Autoimmune haemolytic anaemia (AIHA)

  3. Drug-induced immune haemolytic anaemia (DIIHA)

Explain the difference between alloimmune, autoimmune, and drug-induced immune haemolytic anaemia.

• Alloimmune: Antibodies are directed against foreign RBC antigens, typically from transfusions or pregnancy.

• Autoimmune (AIHA): Antibodies (autoantibodies) are directed against the individual’s own RBCs.

• Drug-induced (DIIHA): Drugs trigger immune responses that lead to RBC destruction via antibodies or immune complexes.

What are autoantibodies, and how can they contribute to shortened RBC survival?

• Autoantibodies are antibodies directed against a person’s own RBCs.

• They can shorten RBC survival by coating RBCs, leading to immune-mediated destruction (via phagocytosis or complement-mediated lysis).

Describe why a positive Direct Antiglobulin Test (DAT) or autocontrol does not always indicate AIHA.

• Positive DAT/autocontrol can be seen in:

  • ~0.1% of normal blood donors

  • Up to 15% of hospitalized patients without haemolysis

• Therefore, a positive test alone does not confirm AIHA; clinical evidence of haemolysis is also required.

Differentiate between compensated and uncompensated anaemia in immune-mediated RBC destruction.

• Compensated anaemia: RBC production ≈ RBC destruction → haemoglobin may remain normal.

• Uncompensated anaemia: RBC destruction > RBC production → haemoglobin decreases, causing anaemia.

List the key serologic tests used to diagnose AIHA.

• Direct antiglobulin test (DAT) with polyspecific and monospecific reagents

• Antibody detection and identification in serum or eluate

How are autoantibodies classified based on temperature, and what is the approximate prevalence?

• Warm autoantibodies: Optimal at 30–37°C (~70% of cases)

• Cold autoantibodies: Optimal at 4–30°C (~18% of cases)

• Drug-induced autoantibodies: ~12% of cases

Describe the clinical significance of cold reactive autoantibodies and their typical titer.

• Often not clinically significant

• Typically tested at 4°C

• Low titer (<1:64 at 4°C)

• Can interfere with ABO typing, antibody screening, DAT, and compatibility testing

Identify the main specificities of cold reactive autoantibodies and explain their relevance.

• Anti-I: Most common; I antigen fully expressed on adult RBCs

• Anti-i: Rare; i antigen expressed on cord RBCs

• Anti-H / Anti-IH: Found in group A1, A1B, occasionally B individuals; reacts best with cells with high H antigen (O, A2)

Compare CHD and PCH.

Feature	Cold Haemagglutinin Disease (CHD)	Paroxysmal Cold Haemoglobinuria (PCH)

Prevalence

Chronic, mainly older adults

Rare (1–2%), mainly children

Antibody

Cold autoantibody (IgM)

Donath-Landsteiner antibody (anti-P)

Clinical

Mild-moderate HA; often asymptomatic

Acute hemolysis, often post-viral

Lab

Reticulocytosis, +DAT, RBC agglutination

Detection of Donath-Landsteiner antibody

Management

Avoid cold, keep warm

Usually supportive, treat underlying infection

Describe the immunoglobulin and complement patterns in WAIHA.

• IgG ± complement (C3) on RBCs

  • IgG + C3: 67%

  • IgG only: 20%

  • C3 only: 13%

Explain the primary mechanism of RBC destruction in WAIHA.

Extravascular hemolysis: IgG-coated RBCs are recognized by macrophages in the spleen and liver → phagocytosis

Outline the three mechanisms of DIIHA.

1. Drug-adsorption (Hapten)

2. Immune complex (“Innocent bystander”)

3. Membrane modification (Non-immunologic protein adsorption)

Explain the drug-adsorption (hapten) mechanism and the most common drug.

• Drug binds covalently to RBC membrane → antibody targets the drug → RBC destruction

• Mostly IgG-mediated

• Most common drug: Penicillin

Describe the immune complex (“innocent bystander”) mechanism and its laboratory confirmation.

• Drug-antibody complex forms in serum → binds RBC → complement activation → RBC lysis

• Lab confirmation: Antibody reacts with RBC only when the drug is added to the test system

What is meant by membrane modification (non-immunologic protein adsorption) mechanism in DIIHA?

Drug modifies RBC membrane → non-specific binding of plasma proteins (IgG, IgM, IgA, complement) → may mimic AIHA without true immune specificity