*ABO and Rh

ISOIMMUNISATION
in Pregnancy

Corina Cardaniuc
Department of obstetrics and
gynecology
 ABO Blood System:
*Four basic blood types ( A, B, AB, and O).
*Each blood type is additionally classified according to the
presence or absence of the Rh factor
 *Blood groups (1900):
Antigens:

O (45%)

A (40%)

B (10%)

AB (5%)

A and B : dominant

O : recessive
 * Rh factor

*The Rh System is a “gene *85% - Rh positive

complex” (Cc, Dd, Ee, Cu, 35% homozigous
cw, Go, I) that reside on
the distal end of the short 50% heterozigous
arm of chromosome one.
*15% - Rh negative
*Rh system is defined by the
presence or absence of D
antigen, a protein found
on the surface of red blood
cells.
 *Rh factor
*RH antigen - genetically determined protein produced
during Red Cells formation.

*It appears as early as the 38 days after fertilization.

*First demonstrated by testing human blood with rabbit

anti sera against red cells of Rhesus monkey and
classifying Rh negative & Rh positive.

*D antigen is primarily responsible for antigenic

behavior and most frequently incriminated in
isoimmunization (97% of all cases).
*

*Rhesus subtypes E, C, e, c
*ABO
*Kell
*Duffey
*Platelets
 * Rh- Isoimunization. Definition

*Rh incompatibility, also Terminology:

known as Rh disease, is a *Rh Disease
condition that occurs
when a woman with Rh- *Alloimmunization
negative blood type is *Isoimmunization
exposed to Rh-positive *HDFN
blood cells of the foetus,
leading to the
*Erythroblastosis Fetalis
development of Rh
antibodies.
 *Incidence
*Literature data show a *Deaths from fetal
frequency of hemolytic hemolytic disease
disease of the newborn to represents about 4% of all
5% (including the ABO perinatal mortality.
system incompatibility).
 * Risck factors for Rh isoimmunization
*Rh negative pregnant women with Rh-positive husband:
the husband phenotype and genotype – homozygous
*Rh is transmitted from the paternal, the autosomal
dominant, so the baby will be Rh father.
*The only problematic situation is when the mother is Rh
negative and the father Rh positive and Rh positive fetus.
*Rh incompatibility doesn't occur in the following situations:
 * Risck factors for Rh isoimmunization
*Normal delivery

Sensitization usually occurs very late in pregnancy,

so the first Rh-positive child is not affected

*Bleeds most often occur at delivery - mother is

sensitized
 * Risck factors for Rh isoimmunization
*Feto-maternal haemorrhage during pregnancy with
leakage of fetal cells in the maternal circulation
Examples:
- Spontaneous abortion
- Induced abortion
- Placental abruption
- Placenta accreta
- Placenta praevia
- Abdominal trauma
- Cordocentesis, amniocentesis
- Ectopic pregnancy
- Caesarean section
- Manual removal or placenta
- Molar pregnancy
-In utero fetal death
 * Risck factors for Rh isoimmunization
*History of Rh isoimmunization (blood transfusion
that contains Rh antigens, hemoterapy, etc)

*ABO compatibility
The protective effect conferred by ABO incompatibility is
believed to be due to maternal destruction and subsequent
clearance of the ABO-incompatible fetal erythrocytes before
Rh sensitization can occur.
 * Risck factors for Rh isoimmunization
*A special case is the isoimmunization of Rh
negative women born from Rh positive mothers,
which can be izoimunizate without any obstetrical
antecedent or transfusion.
* It is assumed that isoimmunization occurs by
transfer of maternal Rh positive blood cells to the
circulation of Rh negative fetus.
* In terms of a pregnancy with Rh positive fetus,
anamnestic reactions develop with significant
increases in antibody titers - the theory of
“troublesome grandmother"
 * Risck factors for Rh isoimmunization

*It is also important to note that once

isoimmunization has occurred,the severity
of sensitization response increases with each
subsequent pregnancy involving a fetus with
Rh-positive blood.
 * The Extend of Severity of Fetal Hemolytic
Diseases:

(1)Volume of fetomaternal hemorrhage.

(2)Degree of maternal immune response
(3)Concurrent ABO incompatibility
 * Estimated Risk of Isoimmunization

If Rhesus prophylaxis is not given:

*Chances of primary sensitization during 1st

pregnancy is only 1-2%
*Second baby - 7% chance of developing antibodies
*Third baby - 17% chance of developing antibodies
*Sixth baby - 30% chance of developing antibodies
 * Pathophysiology
The placenta usually acts as a barrier to fetal blood entering
the maternal circulation.

*Fetalcells can enter the maternal circulation through a

“break” in the “placental barrier”.
 * Pathophysiology
*Antibody formation occurs in two stages:
Sensitisation
Immunisation

*A feto-maternal bleed of 0.1 ml is sufficient for the anti

body production to produce iso immunization
 Cleared by Macrophage
Mother
Memory B lymphocytes
Primary Response

•6 weeks to 6 months

IGM (complete antibodies)

Placenta

High molecular weight

Cannot pass the placenta
Cannot affect the fetus
 Macrophage antigen
Presenting cells
Mother
T- helper cell Seocondary Response

•Small amount
•Rapid - 72 hours
B cell •IgG

Ig G (incomplete antibodies)
Placenta

Fetal Anaemia
 HDN develops IN UTERO

Antigen-Antibody reaction on the RBCs surface  Hemolysis


Anemia

Tissue hypoxia

Hyperplasia of bone marrow release of erythroblasts
“Erythroblastosis fetalis”

Hepatic and splenic erythropoesis
 
Hepatosplenomegaly Hypoproteinemia

Generalized oedema
Ascitis
Polyhydramnios

“Hydrops fetalis”
 After birth

Antigen-Antibody reaction on the RBCs

surface


Hemolysis

Anemia … Jaundice …
Kernicterus


Neonatal death
 *Bilirubin
*Hemoglobin is
metabolized to bilirubin
*Before birth, “indirect”
bilirubin is transported
across placenta and
conjugated in maternal
liver (“direct”) where it
is excreted
 *Bilirubin
* After birth, the newborn liver is
unable to conjugate the bilirubin

* Unconjugated (“indirect”) bilirubin

can reach toxic levels

kernicterus

permanent brain damage

neurological and mental
disorders

Hyperbilirubinemia becomes
apparent only in the delivered
newborn in the absence of
placental clearance
 * Types of fetal hemolytic diseases
*Congenital anemia of new born: It is the mildest form of
the disease where hemolysis is going on slowly.

*Icterus gravis Neonatorum: jaundice develops soon after

in the absence of placental clearance. Bilirubin crosses the
blood brain barrier to damage the basal nuclei of the brain
producing clinical manifestations of Kernicterus.

*Hydrops fetalis: generalized oedema ascites and

hydrothorax. Fetal death occurs sooner or later due to
cardiac failure.
 * Affection in the mother
*Increased incidence of pre-eclampsia
*Polyhydramnios
*Big size of the baby
*Hypofibrinogenemia due to prolonged retention
of dead fetus
*Maternal syndrome or mirror syndrome with
generalized oedema, proteinurea and pruritis
*Repeated still births
*Repeated abortions
 *Diagnosis and management
The goals in managing the alloimmunized pregnancy are 2-
fold:

* Initially detecting fetal anemia prior to the

occurrence of fetal compromise.

* Minimize fetal morbidity and mortality by correcting

this anemia until fetal lung maturity and delivery can be
achieved.
 * Diagnosis and management
* ABO and Rh testing at 1st visit

Rh negative mother

Paternal Blood Group and Rh

Rh negative Rh positive

No treatment Homozygous or Heterozygous

Maternal antibodies (indirect Comb's test)

Negative Antibodies detected – sensitized

Repeat at 26-28 and 34-36 weeks Repeat every 4 weeks

*Diagnosis
 Prophylactic Management of non sensitized
Pregnancy
During labor
*No fundal pushing in 1st or 2nd stage of labor
*No uterine massage or uterine grasp or squeez in 3rd stage
*Let the placenta to be delivered spontaneous
*A void avulsions of the cord
*Protect the vaginal and perineal wounds and lacerations from
being exposed to the fetal blood spilled from cord
During cesarean section
*Use abdominal packs in the sides of the uterus before opening
the lower segment to prevent spilled blood from the placenta
to inter the peritoneal cavity.
*Let the placenta to be delivered spontaneous using control
cord traction without squeezing the uterus
* Avoid avulsions of the cord
 Prophylactic Management of non sensitized
Pregnancy

*Prophylactic anti D immunoglobulin 300 mcg (1 dose) is

given to all negative non sensitized mothers at 28 weeks
and again at 34 / 36 weeks.

*Another dose of Rh Ig should be given to the mother within

72 hours of delivery (Rh positive infant, even if stillborn)

*Rh Ig attaches to fetal RBCs in maternal circulation and

are removed in maternal spleen; this prevents
alloimmunization by mother

*The standard dose of anti D is 0.3 mg —will eradicate 15

ml of fetal (D-positive) red blood cells or 30 mL of whole
fetal blood.
 Prophylactic Management of non sensitized
Pregnancy
*Indications for prophylaxis
Abruptio placenta
IUD
Abortion
Amniocentesis
Ectopic pregnancy
Chorionic villus sampling
Evacuation of a molar pregnancy
Abdominal trauma
Foetal-maternal hemorrhage

Pregnancy <12 weeks- 50-100mcg Anti D 300mcg Anti D

Pregnancy >12 weeks- 300mcg Anti D

* within 72 hours from the bleeding

Prophylactic Management of non sensitized
Pregnancy
 Prophylactic Management of non sensitized
Pregnancy

Antepartum bleeding

Kleihauer –Betke test

Re-calculate the dose of the anti-D

Kleihauer-Betke acid elution: Quantitates the
number of fetal cells in circulation.

Fetal hemoglobin is resistant to acid and

retain their hemoglobin (appear bright pink).

The dose should be doubled or tripled if fetal

RBCs are more than 80 cells in maternal
circulation (10 mcg/1mL fetal blood)
 *
Step 1) stain and count the amount
of fetal cells out of 1000 total cells
counted
Step 2) calculate the amount of fetal
blood in cirulation by multiplying % # fetal cells
x100  % of fetal cells
fetal cells by 50 mL total cells (2000)
Step 3) divide mL of fetal blood by
30 (each vial protects against a 30
mL bleed
Step 4) Round the calculated dose
up and add one more vial for safety

% fetal cells x 50
Required dose of RhIg 
30
 Antepartum Alloimmunization Prophylaxis

*98.5% of Rhesus D disease is prevented by event-

stimulated administration of Anti-D

*99.9% of Rhesus D disease can be prevented by routine

administration Anti-D in the 3rd trimester at 28 weeks
 Failure of prophylaxis

Dose too small

Dose too late >72 hours
Patient already immunized but antibody titer too
low for laboratory recognition
Defective immune globulin given
 * Management of Rh sensitized mothers
*Consider invasive at titer of 1:16 or greater
*A titer of 16-32 is significant
*Titres of ≤ 1:32 are managed noninvasively with repeat
antibody titres every every 4 weeks (16-20 weeks) and every
2 weeks (third trimester)
*Titers >1:32 indicates a need for amniocentesis or
cordocentesis
*Titres of ≥ 1: 64 – amniocentesis to be done at intervals of 2
to 3 weeks, interruption of pregnancy
* If antibodies level continuo at the same level and no fetal
compromise … deliver at term
*A score change of more than 10 is considered a significant
change in titer
 * Management of Rh sensitized mothers
*In advanced pregnancy, a sudden drop in titer is a
warning sign and require immediate measures.

*Decrease in antibody level can be either

immunosuppressive effect of pregnancy or massive
transfer of antibodies in the fetal circulation and
their fixation on fetal erythrocytes.
 * Management of Rh sensitized mothers
- Serial ultrasound to assess fetal well being and detect early signs of
fetal anemia and hydrops starting at 18 weeks and repeated every
2-4 weeks.
- The following changes are signs of alarm, which require careful
follow-up of pregnancy:
Placental thickness> 4 cm
ILA > 18
Abdominal circumference
Umbilical vein diameter > 8 mm

*Once hydrops fetalis has developed, the sonographic

evidence includes scalp edema, cardiomegaly,
hepatomegaly, pleural effusion, and ascites, abnormal fetal
posture (Buddha stance)
 * Doppler ultrasonography
Peak systolic middle cerebral artery Doppler velocimetry

*The middle cerebral artery is

examined close to its origin in the
internal carotid artery.

*The risk of anemia is highest in

fetuses with a peak systolic velocity
of 2.5 times the median or higher.

*The recommended interval between

Doppler ultrasound assessment is
between 7-14 days, starting with a
gestational age of 28 weeks in cases
of isoimmunization.
 *Cardiotocography

*A sinusoidal heart rate pattern is an ominous sign of

severe fetal anemia needing immediate intervention.
 *Amniocentesis
*Amniocentesis assesses the status of
the fetus using amniotic fluid:
*Spectrophotometer (350-700 nm)
*Change in optical density (ΔOD)
above the baseline of 450 nm is the
bilirubin measurement - Lily curve

-Amniocentesis: should be performed

under ultrasound guidance if titre
>1:32
- Start from 20-22 weeks, every 2-4
weeks or more frequent if needed
 *Liley’s chart
* The Lily curve is divided into tree zones.
* Zone 1 indicates mild or no disease.
Conservative management with regular
follow up of fetal and maternal conditions
* Fetuses in zone I are usually followed with
amniocentesis every 2-3 weeks.

* Zone 2 indicates intermediate disease.

Fetuses in low zone II are usually followed
by amniocentesis every 1-2 week.

* Zone 3 suggest severely affected fetus.

Fetuses above the middle of zone II may
require intra-uterine transfusion or
delivery.

* Patients with results in zone I or zone II

can be allowed to proceed to term.
* In most cases, patients in the middle of
zone II can progress to 36-38 weeks of
gestation.
 *Cordocentesis
*Cordocentesis takes a sample of umbilical vessel to
obtain blood sample

*Gold standard for detection of fetal anemia and

exact values of fetal Hb

*About 18-20 weeks’ gestation

*Reserved for patients with increased MCA-PSV or

delta OD450
* Management of Sensitized Pregnancy
Two options in intrauterine life

32-34 weeks <32-34 weeks

Steroids
Steroids
Premature delivery and
Intrauterine teatment
intensive care
 * Intra uterine therapy
• Plasmapheresis - removes the circulating antibodies (<1
mcg/ml)

• It is indicated for women with severe isoimmunization, high

titre of antibodies before 20 SG.

• Steps:
* Whole blood is withdrawn from the person.
* The liquid portion or plasma is removed from the blood and
replaced.
* The blood, with all its red and white blood cells, is transfused
back into the person.
 * Intrauterine transfusion
*Removes bilirubin
*Removes sensitized RBCs
*Removes antibody
Indications:
*Amniotic fluid ΔOD is in high zone II or zone III
*Cordocentesis has Hb<10 g/dL or HCT<30%
*Hydrops on ultrasound
From 22-25 weeks, repeated every 3-4 weeks until
delivery at 34 weeks.

Steroid for fetal lung maturation before procedure

 * Intrauterine transfusion
• Intra peritoneal blood transfusion
“O“ rhesus negative Blood

• Through the umbilical vein –Cordocentesis - “O“ rhesus

negative Red Blood Cells
 * Delivery
Criteria for choosing the method of delivery:

• intrauterine condition of the fetus

• fetal presentation
• cervical status
• gestational age

• Emergent delivery of an infant born with hydrops fetalis

should be as nontraumatic as possible.
• It is preferable to caesarean section to avoid fetal stress
 Postpartum testing

At birth

Neonatal (cord) blood

Maternal blood sample
sample

- antibodies by Direct
Comb's test ( DCT )
-antibodies by indirect
Comb's test ( ICT )
- Infant blood group
-fetal red blood cells in
- Infant bilirubin level
maternal circulation
- Infant Hb and Hct level
 *Neonatal treatments
*Phototherapy (after birth) -exposure to artificial or sunlight
to reduce jaundice
*Change unconjugated bilirubin to biliverdin
*May avoid the need for exchange transfusion

*Newborn exchange transfusion

*Involves removing newborn’s RBCs and replacing them
with normal fresh donor cells
*Via umbilical vein
*Corrects anemia
*Group O, D-negative (Maternal blood if possible)
 *Neonatal treatments
Positive Coombs test
Positive Coombs test Elevated bilirubin
Normal bilirubin, Hb Anemia
Ascitis
Hepatosplenomegaly
Jaundice

No treatment

Neonatal exchange transfusion

 * ABO Blood Group Incompatibility
*ABO incompatibilities are the most common cause
of HDN but are less severe
*ABO HDN can occur during the FIRST pregnancy -
prior sensitization is not necessary

Mother type O Child type A or B

 * ABO Blood Group Incompatibility

*Group O individuals have a high titer of IgG anti-A,B

in addition to having IgM anti-A and anti-B
*Natural anti-A and anti-B IgG can cross the placenta
*Fetal RBCs are less developed at birth, so there is less
destruction by maternal antibodies
*Because most of the anti-A and anti-B antibodies are
immunoglobulin M (IgM), which does not cross the
placenta, the fetal hemolysis does not lead to severe
anemia and hydrops.
 * ABO Blood Group Incompatibility
*Never an intrauterine problem
*There is no adequate method of antenatal diagnosis
*No particular antepartum management needs to be
addressed in the setting of ABO incompatibility.
*When delivered, infants may present with mild anemia or
normal hemoglobin levels
*Usually causes neonatal hyperbilirubinemia and jaundice
within 12 to 48 hours after birth

*Only about 10% require therapy

*Phototherapy is sufficient
*Rarely is exchange transfusion needed
 *

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Nov 2008
* ARCBS Guidelines for the use of Rh (D) Immunoglobulin (Anti-D), available at
http://manual.transfusion.com.au/Pregnancy-and-Anti-D/
Anti-D: mechanism of action, kinetics
* Kumpel BM. On the immunologic basis of Rh immune globulin (anti-D) prophylaxis.
Transfusion 2006; 46:1652-1656
* MacKenzie IZ, Roseman F, Findlay J, Thompson K, Jackson E, Scott J, Reed M. The
kinetics of routine antenatal prophylactic intramuscular injections of polyclonal anti-
D immunoglobulin. BJOG. 2006 Jan;113(1):97-101.
* CSL Bioplasma Rh(D) Immunoglobulin VF Product Information; available at
http://www.csl.com.au/docs/603/830/CT36600198E.pdf; accessed 1 Nov 2008
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pregnant RhD-negative women. BJOG. 2003 Jan;110(1):39-45.
 *
* de Silva PM, Knight RC. Serological testing during pregnancy in women given routine
antenatal anti-D Ig prophylaxis. Transf Med. 1997 Dec;7(4):323-4.
Management of alloimmunization
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Thank you
 Prophylactic anti D immunoglobulin

* Standard Dose: i.m. injection: 500 iu = 100 ugm (UK),

1500iu = 300 ugm (USA)

*1500iu = 300 ugm  neutralize 15ml of fetal (D-

positive) red blood cells or 30 mL of whole fetal
blood
*500 iu = 100 ugm  neutralize 5ml (4ml+1ml) of
fetal (D-positive) red blood cells or 10 mL of whole
fetal blood
*4ml = 4x20 f.cells = 80 fetal cells