Rh ISOIMMUNIZATION

‘
Presenter: Dr. LOKENDRA
BATA
MD Resident 2nd year
 Rh isoimmunisation
• Alloimmunization (isoimmunization) is defined
as a production of immune antibodies in an
individual in response to foreign red cell
antigen derived from another individual of the
same species provided, the first one lacks the
antigen.

In population based screening studies Rate of

isoimmunization is approximately 1%.

Cunningham, Williams, Leveno, Bloom,Spong et al ;Williams Obstetrics 24rd Edition

 History
In 1940, Landsteiner and Wiener identified an
antigen present in blood from a rhesus monkey
which stimulates the production of antibody
when injected into rabbits.

There are currently 30 different blood group

systems and 328 red cell antigens recognized by
the International Society of Blood Transfusion.
Cunningham, Williams, Leveno, Bloom,Spong et al ;Williams Obstetrics 24rd Edition
Rhesus factor :
a factor common to the rhesus monkey; those that lack this
factor are Rh negative. 80% of humans are Rh positive.

Incidence of Rh negative:
34% Basques
15% Caucasians
7% African Americans
5% Indians
1% Orientals
Rh antigen:
Are lipoproteins confined to red cell membrane

Rh blood group includes some 35 other antigens in

addition to 5 antigens (CcDEe)

In Rh-positive persons, 45% are homozygous for D,

while 55% are heterozygous.

Rh D is the most immunogenic antigen

 Maternal isoimmunisation...
Among the antigens capable of causing maternal
alloimmunization and fetal hemolytic disease, the Rh
blood group system is the most common.

In particular, the D antigen of the Rh blood group

system (Rh D) is responsible for the most cases of
severe hemolytic disease.
 How does it occur?
2 main mechanisms
1.When an Rh-negative pregnant mother is exposed to
Rh-positive fetal red blood cells (pregnancy or at
delivery)
2. When a Rh-negative female receives Rh-positive
blood transfusion.

Antibody formation occurs in two stages

•Sensitisation(0.1 ml blood)

•Immunisation
 Pathogenesis
Rh +ve fetal cells enter maternal circulation

remain in circulation (life span) until removed by

R.E.S
destroyed & liberate antigen (D)
triggers RBC Ag specific Ab production
(IgM-doesn’t cross placenta due to large molecular
weight)
Subsequent exposure to Ag , previously primed B
cells produce IgG crosses placenta, coat D positive
fetal RBC
fetal RBC hemolysis in spleen
 Isoimmunisation

Takes almost 6 months (initial mechanism is slow)

1st pregnancy is almost always not affected

Re-exposure to the antigen produces a rapid

immunological response usually measured in days
(IgG)
 Potential sensitizing events for rhesus disease

Virtually all cases of Rh(D) alloimmunization occur due

to silent transplacental fetomaternal bleeding

Frequency and volume of spontaneous fetomaternal

hemorrhage increases with advancing pregnancy.

Incidence in 1st trimester: 56%

2nd trimester: 63%
3rd trimester: 71% Ian donald 7th edition
Fetomaternal hemorrhage
Miscarriage(3-5%)
APH
Pregnancy termination( 6- 25%)
Stillbirth
Ectopic(5- 8%)
Invasive in-utero procedure
(amniocentesis, cvs )
Maternal abdominal trauma
External cephalic version
Caesarean section
Manual removal of placenta
• Alloimmunisation is low for the following reasons

– Insufficient transplacental passage of fetal antigens or

maternal antibodies
– Inborn inability to respond to the Rh antigen stimulus
– Maternal fetal ABO incompatibility which leads to rapid
clearance of fetal erythrocytes before they elicit an
immune response
– Variable maternal immune response to the antigen
– Volume of fetal blood entering into the maternal
circulation
– Father’s Rh status and zygosity

Cunningham, Williams, Leveno, Bloom,Spong et al ;Williams Obstetrics 24rd

 Effect on fetus
Mild Cases: antiD (IgG)coated fetal RBC destruction
in RES ↓
anaemia→ congenital anemia of newborn
↓
compensating haemopoiesis by bone marrow. Bone
marrow when exhausted extramedullary
erythropoiesis(overproduction of immature fetal
and neonatal blood cell (erythroblastosis fetalis) ,
causing hepatosplenomegaly.
↓
excess of unconjugated bilirubin → Icterus gravis
neonatorum
 Effect on fetus
Severe Cases:

excessive destruction of fetal RBCs

↓
severe anaemia
↓
hypoxia of the tissues, metabolic acidosis
↓
generalized oedema , ascites due to hypoproteinemia from
anoxic damage to fetal liver
↓ FETAL HYDROPS
cardiac or circulatory failure
↓
IUFD
 Effects in mother

• Increased incidence of preeclampsia

• Polyhydramnios

• Big baby and its hazards

• PPH due to big placenta

 management
• Rh neg non isoimmunised.
• Rh neg isoimmunised
 Rh neg non isoimmunized
• to determine the Rh phenotype of father. If the
father is Rh negative the baby will be Rh negative
the pregnancy should be managed like any other
normal pregnancy without any further testing .
• If the father is Rh positive, there is 50 (father
heterozygous) to 100% (father homozygous)
probability that the fetus will inherit one copy of
the RHD gene and therefore Rh alloimmunization
may occur during pregnancy.
Fernando Arias, Shirish N Daftary, Amarnath G Bhinde; Practical guide to high risk pregnancy and delivery ; 3rd edition
 Detection of Rh alloimmunization
• Rh sensitization may occur before delivery is
about 1%.
• To identify antepartum sensitization,the antibody
screening should be repeated at 20, 24, and28
weeks of gestation. If anti-D antibodies are
detected,the woman has developed Rh
alloimmunization and her management becomes
similar to that of Rh-negative immunized women.
Fernando Arias, Shirish N Daftary, Amarnath G Bhinde; Practical guide to high risk pregnancy

and delivery ; 3rd edition

If the antibody screening does not show
evidence of alloimmunization, the patient
should Routine prophylaxis with a standard
dose anti D Rh Ig Antibody at 28 weeks
pregnancy.
• Postpartumly immediately after delivery and anti-
D immune globulin given when the following
conditions are fulfilled:
1. The infant is Rh positive.
2. The direct Coombs’ test on umbilical cord blood is
negative. This test reveals whether or not the
infant’s red cells are covered by irregular
antibodies.
3. The crossmatch between anti-D immune globulin
and the mother’s red cells is compatible.

Fernando Arias, Shirish N delivery ; 3rd eDaftary, Amarnath G Bhinde; Practical guide to

high risk pregnancy and dition

 Prophylaxis… Dose calculation

Standard dose : to be Given within 72 hours

300 µgm = 1500IU

neutralize 15ml fetal RBC
30 ml fetal blood

Standard dose for > 12weeks,

½ standard dose for < 12weeks
 Antibody detection
Indirect coomb’s test

Serial antibody titers determined at first visit then 20

wk, 24 wks then 28 wks of gestation.
Anti-D immunoglobulin at 28 wks if no antibody seen.
Arias 3rd edition

If ICT NEGATIVE after28 wks further testing not required.

If clinically significant titre, monthly test till 24 wks & 2 wkly
tests till term.
Critical titer : titer associated with a risk for fetal
hydrops, below this titre risk is mild to moderate,it
varies but usually between 1:8 to 1:32.

If critical titer present or exceeds- further assessment

to determine severity of fetal anemia.
Rh-Negative Isoimmunized Women
• first step, the determination of the paternal
and fetal Rh phenotype and genotype.
Paternal Rh phenotype and genotype---
- If the father of the baby is Rh negative the fetus will not be affected
and further tests are unnecessary.
- If the father is Rh positive it is necessary to determine if he is
homozygous or heterozygous for the RHD gene.

Fernando Arias, Shirish N Daftary, Amarnath G Bhinde; Practical guide to high risk pregnancy and delivery ; 3rd edition
 Fetal Rh determination---
• genotype by chorionic villous biopsy (CVS) or
amniocentesis.
• phenotype can be determined by serologic
testing using fetal blood.
• More recently, noninvasive fetal Rh D blood
typing has been performed using cell-free
fetal DNA from maternal plasma.Accuracy is
reported to be as high as 99 to 100 percent.

Cunningham, Williams, Leveno, Bloom,Spong et al ;Williams Obstetrics 24rd Edition

 If pregnancy shows a positive Anti D
Titre
-- An anti-D titer of 1:8 in the first affected pregnancy is
often the cut off value.
-- When the antibody titer is <1:8 intervention is not
needed.
--Titers greater than 1:4 should be considered Rh
alloimmunized.
-- However, the threshold for invasive fetal testing varies
at different institutions and generally is 1:16 or greater
because these titers have been associated with fetal
hydrops.
 When the titer is >1:16

• Amniocentesis
• percutaneous umbilical cord blood sampling
(cordocentesis) should be considered
 Fetal Assessment in the Alloimmunized
Pregnancy
the goals of managing the alloimmunized
pregnancy are mainly ..
1. detection of fetal anemia prior to the
occurrence of fetal compromise
2. After detection-
the goal is to minimize fetal morbidity and
mortality by correcting anemia until fetal
lung maturity then delivery can be
achieved
 Assessment of severity of fetal anemia :

noninvasive
Two-dimensional ultrasound:
to establish the correct gestational age
to guide invasive procedures and monitor fetal
growth and well-being
to detect fetal hydrops (hydrops not observed until
the fetal hemoglobin deficit is at least 7 g/dL below
the mean for gestational age)
 MIDDLE CEREBRAL ARTERY- PEAK SYSTOLIC
VELOCITY
• Mother not put at risk
for worsening
alloimmunization
• Can be used with
alloantibodies
other than RhD,
including anti-Kell
antibodies
 s

A: moderate to severe anemia; B: mild anemia; C: no anemia; MCA: middle cerebral artery;
MOM: multiples of the median.
Middle cerebral artery-peak systolic velocity:
Doppler assessment of the fetal middle cerebral artery
(MCA) peak systolic velocity emerged as the best
tool for predicting fetal anemia in at-risk pregnancies

Principle : anemic fetus preserves oxygen delivery to

the brain by increasing cerebral flow of low viscosity
blood

MCA peak systolic velocity >1.5MoM – mod to severe

affection
 Assessment of severity of fetal
anemia
• Invasive evaluation-----
• Indirect: spectrophotometry (the ΔOD450)
using a specimen of amniotic fluid obtained by
ultrasound-guided amniocentesis.

● Direct: fetal blood studies using a sample

obtained by fetal blood sampling.
D JAMES High Risk Pregnancy - Management Options, 4 Edition
Amniocentesis
Performed under ultrasound guidance if critical titre
Timing: 1st amniocentesis 10 weeks before previous
IUFD, stillbirth
Start from 20-22 weeks, 2-4 weekly or more frequent
if needed
Amniotic fluid analysis :
- fetal Rh genotype, lung maturity
- spectrophotometry analysis of amniotic fluid
Spectral analysis of amniotic fluid
Estimation of bilirubin in amniotic fluid is done by
spectrophotometry

Performed when
-the critical titer reached or
-if there’s previous seriously affected fetus or
infant
 Spectral analysis of amniotic fluid

Optical density of the

liquor containing the
bilirubin pigment is
observed at 250-
700nm wave length

In presence of bilirubin
there is deviation
bulge peaking at
450nm wave length
 LiLey’s Chart
• Delta OD at 450 should be plotted in Liley
chart.[used between 27 to 41 weeks]
• I t has X axis –indicates gestation in weeks
and Y axis about Delta OD.
• It has 3 zones called Low, Mid and High Zone.
• Delta OD may fall either of the zones and
gives approximate time for time of delivery.
 Liley’s chart

For any POG the height

of spectrophotometric
deviation bulge at 450nm
falls within one of the
three zones when
plotted in Liley’s chart
• Zone I (lowest zone): indicate a fetus at very low risk
of severe fetal anemia
• Zone II (middle zone): mild to moderate fetal
hemolysis, but the risk of severe fetal anemia is low –
repeat after 2 wks
• Zone III (upper zone): severe fetal anemia with a
high probability of fetal death within 7 to 10 days
 Low zone
• Like a normal pregnancy deliver at 38 weeks
• Do regular ultrasound and may have to
repeat amniocentesis.
• Fetal well being tests – NST, Biophysical
profile, Doppler study.
 High mild and high zone
High mid and High zone will require cordocentsis –
to assess fetal hemoglobin, hematocrit , platelets
and group , reticulocyte count fetal transfusion
through umbilical vein and delivery.
Transfusion of O negative fresh blood if
hematocrit is less than 30%.
• High mid zone High Zone – Deliver at 34
weeks electively by cesarean section .
• Inform the neonatologist prior to the delivery.
• Higher tertiary centers is ideal place for
delivery.
 Cordocentesis
(usg guided fetal blood sampling)
The main indication
- MCA PSV > 1.5
MoMs above the mean - -
an amniotic fluid bilirubin
values in the high middle
•Blood grp, DCT zone or in zone 3.
•Hb, reticulocyte count - fetal hydrops by
•Biliribin level
ultrasonographic
Intrauterine transfusion:

• Intraperitoneal
• Intravascular
Indication-
fetus with gestational age<34 wk,
fetal Hb<9gm% or HCT<30%

•Blood transfused- O negative packed cells cross

matched with maternal blood
 Obstetric management
Plan of delivery

Unimmunized mother Immunized mother

Wait till term decision of delivery

then terminate depends on severity
Not beyond EDD
 Immunized mother
Shift to equipped center
neonatal intensive care unit
expert neonatologist
facility of exchange transfusion

When to deliver
Mild cases : 37 -38 weeks

Severe cases:
Sudden rise in titre or high titre
USG features of fetal affection
Raised MCA peak flow velocimetry (>1.5MoM)
Zone III
 Severe cases

>34wks or <34 wks or

Amn fluid study showing Lung immature
fetal lung maturity
Steroid
Termination of preg Intrauterine
exchange transfusion
 Care during delivery
• Prophylactic ergometrine for active management of
3rd stage should be withheld
• Avoid spillage of blood into the peritoneal cavity
during LSCS
• Avoid routine manual removal of placenta
• Early cord clamping
• Cord kept long (15-20cm)
 Postnatal management
The neonate:
Cord blood inv:
Clotted blood: direct coomb’s test, Bl.gp, Rh type,
bilirubin
Anti coagulated blood: Hb, Hct

Mild cases: phototherapy - correction of acidosis

Severe cases : exchange transfusion

Phototherapy (bil > 12mg%)
• Blue or blue green light of 420-470nm wavelength
used
• Degrades bilirubin by photo-oxidation and structural
isomerisation
• Bilirubin converted to water soluble less toxic polar
isomer excreted in bile and urine
 Exchange transfusion
Indications:
1. Rh positive babies with direct coomb’s test positive
2. Cord hemoglobin<10gm/dl
3. Cord Bilirubin level >5mg/dl
4. Rising rate of bilirubin > 1mg/dl/hr despite phototherapy
5. Bilirubin >20mg/dl
6. Previous definite history of an affected baby
7. Birth wt. <2.5kg/ prematurity
Prophylaxis
 Prophylaxis

ANTI-D immune globulin

sterile solution containing IgG anti-D (anti-Rh) manufactured
from human plasma

Anti D (RhD IgG/ RhoGAM) given if baby is Rh positive and

direct Coomb’s test is negative
Following a sensitising event (delivery or other)
Standard dose (300µgm) for > 12weeks and 50µgm for
<12weeks I.M.
Given within 72hours of the incident
Routine administration of anti D during pregnancy:
Standard dose at 28weeks will reduce Rh isoimmunization (from
1.6% to 0.07%)
 References
• Cunningham, Williams, Leveno, Bloom,Spong
et al ;Williams Obstetrics 24rd Edition
• Fernando Arias, Shirish N Daftary, Amarnath G
Bhinde; Practical guide to high risk pregnancy
and delivery ; 3rd edition
• D JAMES High Risk Pregnancy - Management
Options, 4 Edition
• Ian Donald’s Practical obstetric problems 7th
edition
Thank you