T7 - Haemolytic Disease of the Newborn

Question 1

What is HDN characterised by?

Answer 1

Jaundice
- significantly elevated (> 300 µM) and/or rapidly rising (> 8.5 µM/hr) serum bilirubin within 24 hours or > 14 days post partum
Positive DAT
Maternal antibody

Question 2

Kernicterus and Hydrops Fetalis

Answer 2

Severe HDN can lead to kernicterus or hydrops fetalis

1. Kernicterus
   - bilirubin accumulation in the brain, can cause brain damage
2. Hydrops fetalis
   - edema in the foetus, can => death from heart failure

Question 3

Mechanisms of HDN

Answer 3

1. Ag neg mother carrying ag pos child (Ag pos father)
2. Mother is exposed to child RBCs
3. Mother’s immune system mounts immune response against foreign antigen => IgG aby produced
4. During subsequent pregnancy, maternal IgG aby crosses the placenta and enters foetal circulation
5. Maternal IgG aby binds to foetal RBCs (if Ag pos)
6. Aby coated foetal RBCs destroyed by foetal reticuloendothelial system

Question 4

Which Antibodies cause HDN?

Answer 4

IgG antibodies reactive in the IAT
Rhesus
- anti –D, -c, -C, -e, -E
Kell
- anti –K, -k
Duffy
- anti-Fya
MNS, Kidd
ABO

Question 5

Current Antenatal and Postnatal Practice

Answer 5

First trimester visit
ABO and Rh(D) typing
- re-test at 28 weeks
Antibody screen
- if neg re-test at 28 weeks
- if +ve:
- perform Aby ID, determine its clinical significance
- clinical history
- find paternal genotype/phenotype
- foetal genotype/phenotype
- aby quantitation/titration

Question 6

Antibody Titration

Answer 6

Dilutions of plasma are reacted against cells with homozygous expression of the antigen being tested
The titre is the reciprocal of the highest dilution with 1+ reactivity
Titre is clinically significant if ≥ 32 or rises by 2 dilutions

Question 7

Tests for Foetal Anaemia

Answer 7

Performed if quantitation/titration suggests risk of HDN
Historical
- amniocentesis
- cordocentesis
Middle cerebral artery (MCA) doppler ultrasound
- non-invasive
- velocity of blood flow in middle cerebral artery is measured
- increased blood flow = anaemia

Question 8

Quantifying Fetomaternal Haemorrhage - Acid Elution Test

Answer 8

Better known as the Kleihauer Test or Kleihauer-Betke Test
Adult haemoglobin (HbA) is sensitive to acid, while foetal haemoglobin (HbF) is resistant
Expose blood film to acid (denatures and removes HbA, while HbF remains intact within foetal RBCs), stain w/ erythrosine or eosin
Controls
- neg: adult blood
- pos: cord blood diluted 1:100 in adult blood

Question 9

Kleihauer Test Procedure

Answer 9

Ensure there are 100 adult cells in a 40x FOV
Count number of foetal RBCs within at least 10k maternal cells (5-10 40x FOV)
Calculate fetomaternal bleed, based on following assumptions:
- foetal RBCs are 22% larger than adult RBCs
- only 92% of foetal RBCs are stained
- maternal RBC volume is 1800 mL
Foetal RBCs (mL) = (Foetal Cells per FOV / Adult Cells per FOV) x 2400

Question 10

Quantifying Fetomaternal Haemorrhage - Flow Cytometry

Answer 10

Detect D pos cells in maternal blood using fluorescently-labelled anti-D antibody
Controls
- neg: adult Rh(D) neg cells
- pos: 0.25% cord Rh(D) pos cells in adult Rh(D) neg cells
Calculate fetomaternal bleed, based on following assumptions:
- foetal RBCs are 22% larger than adult RBCs
- maternal RBC volume is 1800 mL
Foetal RBCs (mL) = % foetal cells x 21.96

Question 11

HDN Treatment

Answer 11

Phototherapy
- expose newborn to blue light (λ = 460 nm)
- light breaks down bilirubin
- can be as effective as exchange transfusions
Exchange Transfusions
- exchange foetal blood to remove aby and replace RBCs
- rarely performed in newborns
- performed in utero to allow foetal lungs to develop

Question 12

Rh(D) HDN

Answer 12

Before the 1960’s, maternal and foetal Rh(D) incompatibilities were the most common cause of HDN
Recognised that HDN was less severe when mother was Rh(D) AND ABO incompatible (vs Rh(D) incompatible alone)
- anti-A and anti-B aby’s clear Rh(D)pos cells before D ag is recognised by the mother’s immune system
Lead to administration of Rh(D)immunoglobulin in Rh(D)neg mothers => markedly decreased incidence of Rh(D) HDN

Question 13

Rh(D) HDN - Source of anti-D

Answer 13

Prophylactic Rh(D)Ig is isolated from the plasma of Rh(D) neg individuals who produce anti-D

Question 14

Rh(D)Ig Administration

Answer 14

Rh(D)Ig is administered intramuscularly (IM) or intravenously (IV)
IM only
- Rh(D) Immunoglobulin-VF
- 250IU and 625IU are available
IV or IM
- rhophylac
- 1500 IU

Question 15

Prophylaxis for Rh(D) HDN

Answer 15

Only for Rh(D) neg mothers w/o immune production of anti-D
28 weeks
- aby screen (even if initial screen was neg)
- administer Rh(D)Ig AFTER collection of sample for aby screen
34 weeks
- Rh(D)Ig is administered again
Delivery
- administer Rh(D)Ig if newborn is Rh(D)+ve

Question 16

Prophylaxis for Rh(D) HDN - How much anti-D is Administered?

Answer 16

100 U of Rh(D)Ig protects against 1 mL foetal RBCs
Prophylaxis
- 625 IU @ 28 and 34 weeks, and delivery
- more is administered if FMH is > 6 mL
Other sensitising events
- e.g. miscarriage, abortion, amniocentesis, chorionic villus sampling, abdominal trauma
- first trimester - 250 IU
- second and third trimesters - 625 IU
- administer within 72 hours

Question 17

Prophylactic anti-D in the Lab

Answer 17

Can’t be distinguished from immune anti-D
Clinical history is crucial!
If anti-D quant is low and falling, probably prophylactic; if anti-D quant is stable, probably immune
Treatment w/ prophylactic anti-D should continue until it’s confirmed that anti-D is immune