RH isoimmunization and

Anti – D prophylaxis
DR. SHRINIVAS GADAPPA (Prof and HOD)
DR. SWATI BADGIRE (Assistant professor)
Rh-Isoimmunization
Isoimmunization
A process by which immune antibodies are produced in a person by the
entry of an antigen of another individual of same species, the former lacking
the antigen
Rh isoimmunization (Rh incompatibility, Rhesus disease, RhD Hemolytic
Disease of the Newborn)
When Rh– mother gets pregnant to Rh+ fetus —she may be sensitized to Rh
antigen and develop antibodies
These will cross the placenta and cause hemolysis of fetal red blood cells
Rh – isoimmunization occurs
Mismatched blood transfusion
Rh – negative women bearing Rh – positive fetus with feto-maternal
hemorrhage

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 Rh Incompatibility: Pathogenesis
When Mother is Rh-ve, Father is Rh+ve and Baby is Rh+ve
The fetal RBCs are separated from the mother’s circulation by the
layer of cells in the placenta called trophoblast.
However during late pregnancy and especially during childbirth
the fetal RBCs may escape into mother’s circulation – Feto-
maternal Haemorrhage (FMH)
Once these cells reach the Rh – ve mother’s circulation, they are
perceived as an antigen and thus can provoke an antibody
response
Initial response is forming IgM antibodies for short period (6wks – 6
months)
Followed by production of IgG

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 HEMOLYTIC DISEASES OF NEWBORN

Antibodies to fetal RBCs are not usually made before

first childbirth (1st Rh –ve child unaffected)
Repeated pregnancies provoke high IgG antibody levels
in the mother
Maternal IgG antibodies can cross the placenta and
reach the fetal circulation in subsequent pregnancies
If the fetus in subsequent pregnancy is Rh +ve, these
maternal IgG antibodies may adhere to the D antigen on
fetal RBC surface, and it will destroy fetal RBCs leading
to hemolysis.

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 RISK OF ISOIMMUNIZATION

The risk of postpartum immunization increases with the

amount of FMH

A secondary immune response can be stimulated by as little

as 0.05 ml of Rh+ve cells

The risk is about 16% for the first Rh+ABO compatible

pregnancy

The risk is 2-3% in an ABO incompatible pregnancy due to

rapid clearance of fetal cells by destruction with maternal
anti A or anti B antibodies

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 Fetomaternal Hemorrhage (FMH)
FMH leads to sensitization
of Rh D-negative mothers,
resulting in an increased
risk of hemolytic disease
of the newborn
Such events causing FMH
are called as Sensitizing
Events
FMH leading to Rh
isoimmunization:
•6.7% in 1st Trimester
•13.9% in 2nd Trimester
•29% in 3rd Trimester
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Procedures during pregnancy Clinical complications
Amniocentesis, •Threatened abortion,
Umbilical blood sampling\ •Abruptio placentae, and
•Placenta previa
Chorionic villous sampling
•MTP
External Cephalic Version
•Intrauterine fetal death
•Vesicular mole
•Miscarriages
Amount needed for
sensitization 0.1 ml
•0.1 FMH/ml – 1%
sensitization
•0.5 – 1 FMH/ml – 25%
•> 5 FMH/ml – 65%
Hemolytic Disease of the Newborn (HDN)
HDN occurs when the baby’s red blood cells are destroyed.
Hemolytic disease – major cause of perinatal mortality, morbidity
and long term disability
(Cochrane 2015)

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 Complications/Dangers of HDN
Hydrops Fetalis

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Disease Burden: Global

Still birth & Neonatal

death due to Rh disease
high in South Asian
Countries such as India,
Afghanistan

Still birth
due to Rh
Disease

Neonatal Deaths due

to Rh Disease

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Prevalence of Rh negative population in India-
State wise

Manipur – 5.8%
Arunachal Pradesh – 1%
Meghalaya – 1.23%
West Bengal – 0.03%
Southern Rajasthan – 5.8%
Himachal Pradesh – 10.7%
Kashmir – 8.83%
Uttaranchal – 10.73%
Uttar Pradesh – 4.41%
Chhattisgarh – 3.15%
Madhya Pradesh – 4.6%
Karnataka – 5.35%
Pondicherry – 6.5%
Telangana – 3.82%
Maharashtra – 5%

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Screening and Diagnosis

Maternal blood grouping, Rh-typing and antibody screening at their 1st pre-
natal visit
Presence of anti-D antibodies in serum is diagnostic of maternal Rh-
isoimmunization

Gold Standard Test

Indirect Coombs: (To detect isoimmunization before delivery)
Mix Rh(D)+ cells with maternal serum
Anti-Rh(D) Ab will adhere
RBC’s then washed & suspended in Coombs serum (antihuman globulin)
RBC’s coated with Ab will be agglutinated

Direct Coombs: (To detect isoimmunization after delivery)

Mix infant’s RBC’s with Coombs serum
Maternal Ab present if cells agglutinate

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 INDIRECT COOMB’S TEST
Administration of anti D in antenatal period to prevent isoimmunization

Indirect Coomb’s test is done to check whether the mother is sensitized or not
5% saline suspension of positive red cells + mother’s blood

Incubated for 1 hr at 37oC

Cells washed 3 times in saline

Coomb’s serum added and centrifuged

Checked for agglutination

Agglutination present = ICT + ve = mother is sensitized

Agglutination absent = ICT –ve = mother not sensitized = give Anti – D prophylaxis

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Screening and Diagnosis
USG
Detection of Hydrops Fetalis
Chorionic villous sampling:
To know fetal Rh genotype
Amniocentesis:
To detect amniotic fluid bilirubin
Invasive
Cordocentesis:
To detect fetal anemia
Invasive
Middle Cerebral Artery – Peak Systolic Velocity (MCA – PSV) – Colour
Doppler
Non Invasive
To determine severity of fetal anemia

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Management

Aim of management during pregnancy:

To predict which pregnancy is at risk
To predict whether or not fetus is severely affected
To correct anemia and reverse hydrops by intrauterine
transfusion
To deliver the baby at the appropriate time, weighing the
risks of prematurity against these of intrauterine transfusion
Objectives
Non immunized women – Prevention of allo – immunization
Immunized women – early detection and adequate
treatment of fetal anemia

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Rh – isoimmunization Prevention

Screening of all mothers for blood group

Screening of antibody for Rh negative mother
Administer anti – D immunoglobulin prophylaxis
All Rh negative mothers after delivery if fetus is Rh
positive within 72 hours
After sensitizing events within 72 hours

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Anti D prophylaxis Guidelines
Anti- D is an antibody against D Epitope present on fetal RBC, to prevent the
Rh –ve women from sensitization

Mechanism of action:

Anti D immunoglobulin passive injection – coats the “D” epitope and

prevent the interaction of maternal immune system with Rh +ve

300 μg anti-D neutralizes 30 mL fetal Rh-positive blood (15 mL packed fetal

RBC)

Available anti-D preparations:

Monoclonal

Polyclonal

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Monoclonal Anti – D immunoglobulin

Highly specific antibodies against

single antigenic epitope produced
by the clone of a single hybrid cell
formed in the laboratory by the
fusion
Advantages
Specific
Consistent
Uniform
Safe from transmission of viruses
and harmful proteins

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 FEATURES ADVANTAGES BENEFITS

Manufactured by Provides monoclonal anti Higher specificity of

hybridoma technology D action

No pooled plasma No batch to batch No shortages unlike

involved in variation polyclonal anti D
manufacturing process
Unlimited production

No risk from emerging Undoubted safety

diseases like CJD, Uniformity of product
Parvovirus infection with competitive pricing
Superior technology

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Polyclonal Anti – D immunoglobulin

Polyclonal-derived from sera of immunized humans, made up

of mixture of antibodies with affinity for a wide range of
antigenic epitopes
Difficult to manufacture in large quantities
Difficult to provide consistent batches
Chances of transmission of infection and harmful proteins

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Comparison between Monoclonal and Polyclonal anti-D
antibodies

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 Monoclonal anti - D immunoglobulin
efficacy testing
Binding – The number of anti-D antibody molecules bound on the
surface of one D+ve RBC.

The amount of Monoclonal anti - D immunoglobulin bound to the surface of D

cells was same as that of polyclonal antibody, indicating comparable binding
efficiency and affinity as that of polyclonal preparation.

ADCC (antibody dependant cellular cytotoxicity) – The amount of lysis

of sensitized RBCs at different concentrations of anti D.

Monoclonal anti - D immunoglobulin found highly active in ADCC assay

Rate of red cell clearance – The ability of Monoclonal anti - D

immunoglobulin to clear the D positive RBCs through the spleen.

The study proves that specificity for Monoclonal anti - D immunoglobulin is

only for sensitized cells and not for non sensitized RBCs. There is complete
clearance of sensitized RBCs from spleen within 3 hrs of administration of
Monoclonal anti - D immunoglobulin

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 Monoclonal anti - D
immunoglobulin
Phase III Clinical Trials

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 Monoclonal anti - D immunoglobulin
clinical trials
The study was carried out to confirm the safety & efficacy of
Monoclonal anti - D immunoglobulin for the prevention of
sensitization of Rh negative mothers
The trial was conducted on 100 patients in LTMG hospital,
Mumbai
Titers were taken on days 3, 30, 75 & 180
Results were evaluated with ICT
Titers were negative upto day 180
Monoclonal anti - D immunoglobulin is effective in prevention of
sensitization
Published in Obs & Gynae Today Aug 2002

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 Monoclonal anti - D immunoglobulin clinical
trials
The study was carried out to compare the safety & efficacy of Monoclonal anti - D
immunoglobulin with polyclonal anti D for the prevention of sensitization of Rh
negative mothers
The trial was conducted in 140 patients in Seth G S Medical College (KEM hospital),
Mumbai
Group 1 consists of 80 patients – all given Monoclonal anti - D immunoglobulin
Group 2 consists of 60 patients – 30 given Monoclonal anti - D immunoglobulin &
30 given polyclonal antiD
Titers were taken on days 3, 30, 75 & 180
Results were evaluated with ICT
Titers were negative upto day 180
Monoclonal anti - D immunoglobulin is more effective in prevention of sensitization
Published in JAPI, Journal of Association of Physicians of India, October 2002

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 ANTI- D IMMUNOGLOBULIN PROPHYLAXIS

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ANTENATL ANTI-D PROPHYLAXIS (AADP)

First Trimester events:

•Significant bleeding during
threatened abortion
•Spontaneous miscarriage In all these events a
•Medical termination of pregnancy dose of 50-100µg
•Surgical termination of pregnancy anti-D Ig given, as
•Ectopic pregnancy soon as possible
•Vesicular mole; particularly of it is after sensitizing
a partial mole
•Chorion biopsy
event
•Embryo reduction

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Routine antenatal anti-D prophylaxis
Routine antenatal prophylaxis reduces the rate of
sensitization during pregnancy to 0.2 %.
Without antenatal anti-D prophylaxis, 1.6 % to 1.9 % of Rh-
negative women at risk become sensitized.
All pregnant women (D-negative or D-positive) should be
typed and screened for alloantibodies, with an indirect
antiglobulin test at the first prenatal visit and again at
weeks. (III-C)
Rh testing of the baby’s father may be offered to all Rh-
negative pregnant women to eliminate unnecessary blood
product administration.(III-C)

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 Anti-D Ig 300µg should be given routinely to all Rh-
negative non-sensitized women at 28 weeks gestation
when fetal blood type is unknown or known to be Rh-
positive.
A repeat antepartum dose of Rh-immune globulin is
generally not required at 40 weeks, provided that the
antepartum injection was given no earlier than 28 weeks
gestation.

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Guidelines recommendations for anti – D
prophylaxis
  ACOG RCOG FOGSI BCSH NHS SOGC RANZCOG
Postpartum 300ug within 300ug within 300ug within 300ug within 300ug within 300ug within 300ug within 72
Prophylaxis 72 hours of 72 hours of 72 hours of 72 hours of 72 hours of 72 hours of hours of delivery
delivery delivery delivery delivery delivery delivery
Sensitizing Events <12 weeks 50- Within 72 50-100ug Within 72 500 IU <12 weeks – First trimester
120ug; >12 hours hours 120ug 250 IU
week 300ug < 12 weeks < 12 weeks >12 weeks Second and third
only for only for ectopic 300ug trimester 625 IU
ectopic pregnancy,
pregnancy, molar
molar pregnancy,
pregnancy, termination of
termination pregnancy,
of pregnancy, heavy uterine
heavy uterine bleeding with
bleeding with pain; 250 IU
pain; 250 IU 12-20 weeks –
12-20 weeks 250 IU
– 250 IU >20 weeks –
>20 weeks – 500 IU
500 IU

ACOG- American College of Obstetrics and Gynecology; RCOG – Royal College of Obstetrics and Gynecology; FOGSI – Federation of Obstetrics and Gynecology Society
of India; BCSH – British Society of Hematology; NHS – National Health Science; SOGC – Society of Obstetrics and Gynecology, Canada; RANZCOG – Royal Australia and
New Zealand College of Obstetrics and Gynecology

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MANAGEMENT OF AN Rh ISOIMMUNISED
PREGNANCY

An antibody titre greater than 1:4 indicates Rh

isoimmunization

If initial anti D titre is less than the critical titre, the patient is
kept under observation with monthly anti D titres from 16 to
18 weeks onwards.

Amniocentesis performed only if titre reaches the critical value

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 Contd.
The maternal antibodies are measured at 2 to 3 weekly
intervals from 17th week

When the antibody level is less than 4 IU/ml, the fetal

haemolysis is mild and the pregnancy can be allowed till term
with a spontaneous delivery

When antibody level between 4 and 8 IU/ml, there is moderate

haemolysis warranting delivery at 38 weeks with a neonatal
transfusion being required in 55%

When antibody levels more than 10 IU/ml, the disease may be

severe and needs further investigation

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Antenatal
Anti D prophylaxis

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Summary
Prevalence of Rh negative population range 5-15%
Rh negative mothers bearing Rh positive fetus has high chance of
developing antibodies against fetal RBCs
Rh positive fetus or babies born to sensitized Rh negative mothers
have high risk of developing hemolytic disease of newborn
Sensitization in Rh negative mothers can be prevented by giving
anti – D prophylaxis in the postpartum period and sensitizing
events such as abortions
Monoclonal anti D preparations offer advantages of specificity,
uniformity, consistency and safety over polyclonal
Management of sensitized Rh negative pregnancy requires frequent
monitoring and expertise of intrauterine blood transfusion

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 THANK YOU

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