Rhesus Isoimmunisation

Rhesus (Rh) isoimmunisation is the maternal immune response against fetal red cell antigens (chiefly Rh-D) that the mother lacks, leading to haemolytic disease of the fetus and newborn (HDFN). It remains a favourite NEET PG topic because the entire chain — sensitisation mechanism, MCA Doppler, intrauterine transfusion (IUT), and the dose and timing of anti-D — is rich in cut-offs and named criteria.

Definition & basic immunology

Isoimmunisation (alloimmunisation) = development of maternal antibodies against fetal RBC antigens inherited from the father but absent in the mother. The clinical syndrome produced in the fetus/newborn is erythroblastosis fetalis / HDFN.

The Rhesus system has 5 major antigens — C, c, D, E, e (there is no "d" antigen). The D antigen is by far the most immunogenic and the usual culprit. An Rh-negative individual lacks the D antigen (genotype dd). Rh isoimmunisation classically requires:

1. The fetus is Rh-D positive (father contributes D).
2. The mother is Rh-D negative.
3. A feto-maternal haemorrhage (FMH) crosses red cells into maternal circulation.
4. The mother is immunocompetent and mounts an antibody response.

High-yield: The first incompatible pregnancy usually only sensitises the mother (primary IgM response, which does not cross the placenta). The IgG anti-D produced in subsequent pregnancies crosses the placenta and causes haemolysis — hence the disease typically worsens with each successive Rh-positive pregnancy.

Why ABO incompatibility partly protects

If the mother is also ABO-incompatible with the fetus (e.g., mother O, fetus A), fetal cells entering maternal blood are rapidly destroyed by anti-A/anti-B before they can sensitise against D. Thus ABO incompatibility offers partial protection against Rh sensitisation.

Etiology — events causing feto-maternal haemorrhage

Sensitisation needs fetal RBCs in the maternal circulation. The volume needed to sensitise can be as small as 0.1 mL.

Sensitising event	Comment
Normal delivery	Largest FMH at the third stage (placental separation)
Caesarean section, manual removal of placenta	Higher FMH volume
Abortion (spontaneous/induced), ectopic	Risk rises with gestational age
Amniocentesis, CVS, cordocentesis	Invasive procedures
External cephalic version	Trauma to placenta
Antepartum haemorrhage (abruption, praevia)	Significant FMH
Abdominal trauma	Blunt injury
Molar pregnancy	Lower risk (no fetal RBCs in complete mole)

High-yield: Roughly 1–2% of Rh-negative women become sensitised antenatally (silent FMH in the third trimester) even without an obvious event — this is the rationale for routine antenatal anti-D at 28 weeks.

Pathophysiology — from antibody to hydrops

Sensitising event → maternal IgM (primary) → re-exposure → IgG anti-D → crosses placenta → coats fetal D-positive RBCs → extravascular haemolysis in fetal spleen → fetal anaemia.

The fetus compensates by:

• Extramedullary haematopoiesis in liver and spleen → hepatosplenomegaly, portal hypertension.
• Release of immature nucleated RBCs (erythroblasts) → the term erythroblastosis fetalis.

Progressive anaemia → high-output cardiac failure + hypoproteinaemia (hepatic dysfunction reduces albumin) → generalised oedema = hydrops fetalis (ascites, pleural/pericardial effusion, skin oedema, placentomegaly, polyhydramnios). Severe hydrops carries a high risk of intrauterine death.

After birth, the placenta no longer clears bilirubin, so unconjugated bilirubin accumulates rapidly → risk of kernicterus (bilirubin deposition in basal ganglia).

Clinical spectrum

The severity of HDFN forms a continuum:

1. Mild — anaemia + mild neonatal jaundice; ~50% need no treatment.
2. Moderate — significant anaemia + hyperbilirubinaemia; risk of kernicterus.
3. Severe — hydrops fetalis, in-utero or neonatal death.

Neonatal features: pallor, jaundice appearing within first 24 hours (always pathological), hepatosplenomegaly, oedema. Icterus gravis neonatorum and kernicterus are feared sequelae.

Diagnosis & investigations

Maternal evaluation

• Blood group and Rh typing of mother at booking; if Rh-negative, type the father.
• Indirect Coombs test (ICT) — detects free anti-D antibodies in maternal serum; this is the screening test. Repeat as needed.
• Antibody titre: A critical titre of 1:16 (some texts 1:8–1:32) marks the level above which there is significant risk of hydrops and the need to move to fetal surveillance with MCA Doppler.

High-yield: ICT (indirect Coombs) screens the mother's serum; DCT (direct Coombs) is done on neonatal/cord blood to detect antibody already coating fetal RBCs. DCT positive = HDFN confirmed in the newborn.

Father and fetal genotyping

• If the father is homozygous (DD), all offspring are Rh-positive; if heterozygous (Dd), 50% chance.
• Cell-free fetal DNA (cffDNA) from maternal plasma can non-invasively determine fetal RhD status from the late first trimester onward — increasingly the modern first step.

Assessing fetal anaemia — the evolution of methods

Method	Use today	Key point
Amniotic fluid ΔOD₄₅₀ (Liley chart)	Largely historical	Measures bilirubin in liquor at 450 nm; needs amniocentesis
MCA peak systolic velocity (PSV) Doppler	Investigation of choice	Non-invasive; detects fetal anaemia
Cordocentesis (fetal blood sampling)	Gold standard for Hb, but invasive	Used at time of IUT

Liley chart (know it for exams)

The Liley chart plots amniotic fluid ΔOD₄₅₀ (a measure of bilirubin) against gestational age (valid after 27 weeks), divided into three zones:

• Zone 1 (lower) — unaffected/mildly affected fetus.
• Zone 2 (middle) — moderate disease; repeat testing.
• Zone 3 (upper) — severe anaemia/hydrops → immediate intervention (IUT or delivery).

The Queenan chart is an extension valid from 14 weeks onward. Both are now largely replaced by Doppler.

MCA-PSV Doppler — the modern cornerstone

Anaemic fetuses have reduced blood viscosity and a hyperdynamic circulation, so blood flows faster in the middle cerebral artery.

High-yield: An MCA-PSV >1.5 MoM (multiples of the median for gestational age) predicts moderate-to-severe fetal anaemia with ~100% sensitivity. This is the trigger for cordocentesis ± intrauterine transfusion. MCA Doppler has replaced serial amniocentesis.

Other ultrasound signs of established disease: ascites, skin oedema, pericardial effusion, hepatosplenomegaly, polyhydramnios, placentomegaly.

Management

Approach in a sensitised pregnancy

Confirm Rh-negative mother + positive ICT → check antibody titre → titre ≥1:16 (critical) → serial MCA-PSV Doppler → PSV >1.5 MoM → cordocentesis to confirm anaemia → intrauterine transfusion if remote from term, or deliver if near term.

Intrauterine transfusion (IUT)

• Indication: fetal anaemia (MCA-PSV >1.5 MoM or fetal Hb low on cordocentesis) when the fetus is too premature for safe delivery (generally <34–35 weeks).
• Blood used: O-negative, Rh-negative, CMV-negative, irradiated, leucodepleted packed cells, cross-matched against maternal serum, packed to a high haematocrit (~75–85%).
• Routes: Intravascular (intraumbilical vein at the cord insertion) is preferred and most effective; intraperitoneal is an alternative.
• Repeated every 1–3 weeks as needed until delivery.

Timing of delivery

Aim to deliver around 37–38 weeks in well-controlled cases (or after a course of antenatal corticosteroids if preterm delivery is planned). Earlier delivery if disease is worsening near viability and IUT is no longer safe.

Neonatal management

• Phototherapy for hyperbilirubinaemia.
• Exchange transfusion for severe hyperbilirubinaemia (risk of kernicterus) or severe anaemia — uses O-negative blood.
• IVIG may reduce the need for exchange transfusion.

Prevention — anti-D immunoglobulin (the most tested area)

Anti-D works by passive immunisation / antibody-mediated immune suppression: injected anti-D coats fetal D-positive cells in maternal circulation and clears them before the maternal immune system is sensitised. It is only useful for prophylaxis in a non-sensitised (ICT-negative) Rh-negative mother — it is useless once the mother is already sensitised.

High-yield: Anti-D is given to a Rh-negative, non-sensitised (ICT-negative) mother carrying (or potentially carrying) an Rh-positive fetus. Once anti-D antibodies are already present (true sensitisation), prophylaxis cannot help.

Standard dosing & timing

Scenario	Dose & timing
Routine antenatal prophylaxis	300 µg (1500 IU) at 28 weeks (± a second dose at 34 weeks in some protocols)
Postnatal (Rh-positive baby)	300 µg within 72 hours of delivery
First-trimester event (abortion, ectopic, CVS, molar)	50 µg (smaller fetal red cell mass)
After amniocentesis / APH / ECV (>12 weeks)	300 µg
Threatened abortion	Consider 50–300 µg per gestation

A standard 300 µg dose neutralises ~30 mL of fetal whole blood (≈15 mL of fetal RBCs).

High-yield (NEET favourite): Postnatal anti-D must be given within 72 hours of delivery. If accidentally missed, it may still confer some benefit if given up to 28 days — so give it anyway.

Kleihauer–Betke test — quantifying large FMH

When a large feto-maternal haemorrhage is suspected (abruption, trauma, manual removal of placenta), a single 300 µg dose may be insufficient.

• Kleihauer–Betke (acid elution) test quantifies fetal RBCs in maternal blood. Fetal haemoglobin (HbF) resists acid elution and stains pink, while maternal cells appear as ghost cells.
• The fetal cell fraction is used to calculate extra anti-D required (each 300 µg covers ~30 mL fetal blood / 15 mL packed fetal cells).
• Flow cytometry is a more accurate modern alternative.

High-yield: Kleihauer–Betke test is indicated to quantify the volume of feto-maternal haemorrhage and thus the dose of anti-D needed — classically after delivery with suspected large FMH, abruption, or trauma. It is not a screening test for sensitisation.

Mnemonic for anti-D dosing situations

"DEATH-AM" — events needing anti-D: Delivery (Rh+ baby), Ectopic, Abortion, Trauma (abdominal), Haemorrhage (antepartum), Amniocentesis/invasive procedures, Molar/Manual removal & Miscarriage. Add ECV (external cephalic version).

Complications

• Hydrops fetalis and intrauterine fetal death.
• Kernicterus (bilirubin encephalopathy) — choreoathetoid cerebral palsy, sensorineural deafness, upward gaze palsy.
• Severe neonatal anaemia and high-output failure.
• IUT-related: cord haematoma, bradycardia, fetal loss, infection.
• Late hyporegenerative anaemia in neonates who had IUT.

Key differentials (non-immune vs immune hydrops)

Always distinguish immune from non-immune hydrops. Causes of non-immune hydrops (now the commonest cause of hydrops overall) include:

Category	Examples
Cardiac	Structural defects, arrhythmias (SVT)
Chromosomal	Turner syndrome, trisomies
Infections (TORCH)	Parvovirus B19 (classic — aplastic anaemia), CMV, syphilis
Haematological	Alpha-thalassaemia major (Bart's hydrops)
Thoracic/twin	CCAM, diaphragmatic hernia, TTTS

High-yield: A negative DCT/ICT with hydrops points to non-immune hydrops — think parvovirus B19 and alpha-thalassaemia first.

Other antibodies can cause HDFN too: anti-Kell (also causes erythroid suppression, not just haemolysis, and MCA Doppler underestimates the anaemia), anti-c, anti-E. Anti-Lewis (Le) and anti-I antibodies are IgM and do NOT cause HDFN (a classic MCQ point).

Recently asked / exam angle

• Timing of anti-D: "within 72 hours of delivery" and "28 weeks antenatal" are repeatedly tested.
• Dose of anti-D: 300 µg standard; 50 µg for first-trimester events.
• Kleihauer–Betke test — indication (quantify FMH/anti-D dose) and principle (HbF resists acid elution; maternal cells = ghost cells).
• MCA-PSV >1.5 MoM as the marker of fetal anaemia and the investigation of choice (replacing amniocentesis/Liley chart).
• ICT vs DCT — which is maternal, which is neonatal.
• Liley chart — three zones, valid after 27 weeks; Queenan from 14 weeks.
• Blood for IUT — O-negative, irradiated, CMV-negative, leucodepleted, cross-matched with mother.
• Anti-Kell disproportionately suppresses erythropoiesis; anti-Lewis does not cause HDFN.
• Anti-D is useless in an already sensitised mother.
• ABO incompatibility is protective against Rh sensitisation.

Rapid revision

1. D is the most immunogenic Rh antigen; there is no "d" antigen.
2. First incompatible pregnancy sensitises (IgM); subsequent ones cause disease (IgG crosses placenta).
3. As little as 0.1 mL of fetal blood can sensitise.
4. ICT = mother's serum (screening); DCT = neonatal cord blood (confirms HDFN).
5. Critical antibody titre = 1:16 → start MCA Doppler surveillance.
6. MCA-PSV >1.5 MoM = moderate–severe fetal anaemia → cordocentesis ± IUT.
7. Liley chart (ΔOD₄₅₀) after 27 weeks; Queenan from 14 weeks; both now historical.
8. Routine antenatal anti-D at 28 weeks; postnatal within 72 hours; first-trimester events = 50 µg, otherwise 300 µg.
9. 300 µg anti-D neutralises ~30 mL fetal whole blood (~15 mL packed cells).
10. Kleihauer–Betke quantifies large FMH to calculate extra anti-D; HbF resists acid elution.
11. IUT blood: O-negative, Rh-negative, CMV-negative, irradiated, leucodepleted, cross-matched with mother; intravascular route preferred.
12. Negative Coombs + hydrops → non-immune hydrops (parvovirus B19, alpha-thalassaemia); anti-Lewis/anti-I do not cause HDFN, anti-Kell suppresses erythropoiesis.