Rh Isoimmunization

Professor Hassan A Nasrat

Chairman of the Department of Obstetrics and Gynecology
Faculty of Medicine
King Abdul-Aziz University

ISO: is a prefix means similar, equal or uniform.

Isoimmunization: is the process of immunizing a
species with antigen derived from the same subject.

Alloimmune Hemolytic Disease Of The Fetus / Newborn:

Definition:
The Red Cells Of The Fetus Or Newborn Are Destroyed By
Maternally Derived Alloantibodies
The Antibodies Arise In The Mother As The Direct Result Of A
Blood Group Incompatibility Between The Mother And Fetus
e.g. When An RhD Negative Mother Carries An RhD Positive
Fetus.
In The Fetus: Erythroblastosis Fetalis
In The Newborn: HDN.

Antibodies That May Be Detected During Pregnancy:

Innocuous Antibodies:
Most Of These Antibody Are IgM Therefore Cannot Cross The Placental Barrier
E.G. Those Directed Against Such Specificities As A, P(1), Le(a), M, I, IH And
Sd(a).

Antibodies Capable Of Causing Significant Hemolytic Transfusion

Reactions:
IgG antibodies, Their Corresponding Antigens Are Not Well Developed At Birth
E.g. Lu (b), Yt (a), And VEL
Antibodies That Are Responsible For HDN :
Anti-c, Anti-d, Anti-e, And Anti-k (Kell)

The RH Antigen Biochemical and Genetic Aspects

Mechanism of Development of Maternal Rh Isoimmunization
Natural History of Maternal isoimmunization /HD of the Newborn
Diagnosis of Rh isoimmunization

The Rh Antigen- Biochemical Aspects:

The Rh Antigen Is A Complex Lipoprotein.

It Has A Molecular Weight Of Approximately 30,000.
It Is Distributed Throughout The Erythrocyte Membrane In
A Nonrandom Fashion.

The Surface Antigens Can Not Be Seen By Routine

Microscopy, But Can Be Identified By Specific Antisera

Function of the Rh antigen:

Its Precise Function Is Unknown.
Rh Null Erythrocytes Have Increased Osmotic Fragility And
Abnormal Shapes.

The RH Antigen- Genetic Aspect

The Rh gene complex is located on the distal end of the

short arm of chromosome one.

A given Rh antigen complex is determined by a specific

gene sequence inherited in a Mendelian fashion from the
parents. one haploid from the mother and one from the
father.

Three genetic loci, determine the Rh antigen (i.e. Rh

blood group).

Each chromosome will be either D positive or D negative

(there is no "d" antigen), C or c positive, and E or e positive.

Genetic Expression (Rh Surface Protein Antigenicity):

Grades Of Positively Due To Variation In The Degree

Genetic Expression Of The D Antigen.
Incomplete Expression May Result In A Weakly Positive
Patient e.g. Du Variant Of Weakly Rh Positive Patient
(They May Even Be Determined As Rh Negative).
A Mother With Du Rh Blood Group (Although Genetically
Positive) May Become Sensitized From A D-positive Fetus
Or The Other Way Around May Take Place.

Genetic Expression (Rh Surface Protein Antigenicity):

Du Variant

Frank D Positive

Incomplete Expression Of The D Antigen Result In A Weakly Positive Patient

e.g. Du Variant Of Weakly Rh Positive Patient.

Factors Affect The Expression Of The Rh Antigen

The Number Of Specific Rh-antigen Sites:

- The Gene Dose,
- The Relative Position Of The Alleles,
- The Presence Or Absence Of Regulator Genes.

Interaction Of Other Components Of The Rh Blood Group.

Erythrocytes Of Individuals Of Genotype Cde/cde Express Less D Antigen
Than Do The Erythrocytes Of Individuals Of Genotype cDE/cde.

The Exposure Of The D Antigen On The Surface Of The

Red Cell Membrane.

Phenotype

Genotype
eCd/EcD

D positive
Antigenicity of the Rh surface
protein:

eCd

genetic expression of the D

allele.
Number of specific Rh
antigen sites.

Ec D

Interaction of components
of the Rh gene complex.
Exposure of the D antigen
on the surface of the red cell

The Mechanism of Development of the Rh Immune Response:

Fetal RBC with Rh +ve antigen
Maternal circulation of an Rh ve mother
The Rh +ve antigen will be cleared by macrophages; processed
and transferred to plasma stem cell precursors (Develop an almost
permanent immunologic memory)

(Primary immune response)

With subsequent exposure the plasma cell line proliferate to produce

humeral antibodies

(Secondary immune response).

The Primary Response:

Is a slow response (6 weeks to 6 months).

IgM antibodies
a molecular weight of 900,000 that does not cross the
placenta.

The Secondary Response:

Is a Rapid response
IgG antibodies
a molecular weight of 160,000 that cross the placenta.

Exposure to maternal antigen in utero the grandmother theory:

This theory explains the development of fetal isoimmunization in a primigravida,
who has no history of exposure to incompatible Rh blood. If a fetus is Rh negative
and the mother is Rh positive, the may be exposed to the maternal Rh antigen
through maternal-fetal transplacental bleed. In such cases the fetus immune
system develop a permanent template (memory) for the Rh-positive antigen.
When the fetus becomes a mother herself and exposed to a new load of D antigen
from her fetus (hence the grandmother connection) the immune memory is
recalled and a secondary immune response occur.

Natural History of Rh Isoimmunization And HD Fetus and Ne

Without treatment:
less than 20% of Rh D incompatible pregnancies
actually lead to maternal isoimmunization

25-30% of the offspring will have some degree of

hemolytic anemia and hyperbilirubinemia.

20-25% will be hydropic and often will die either in

utero or in the neonatal period.

Cases of hemolysis in the newborn that do not

result in fetal hydrops still can lead to kernicterus.

The Risk of development of Fetal Rh-disease is

affected by:
Less than 20% of Rh D incompatible pregnancies actually
lead to maternal alloimmunization

The Husband Phenotype And Genotype (40 % Of

Rh Positive Men Are Homozygous And 60% Are
Heterozygous).

The Antigen Load And Frequency Of Exposure.

ABO Incompatibility

Why Not All the Fetuses of Isoimmunized Women

Develop the Same Degree of Disease?

The Amount Of Fetal Cells In Maternal Blood

The Non-responders:
ABO Incompatibility:
Antigenic Expression Of The Rh Antigen:
Classes Of IgG Family

Diagnosis of Rh isoimmunization

The diagnose is Based on the presence

of anti-Rh (D) antibody in maternal
serum.
Methods of Detecting Anti D Antibodies in
Maternal Serum:

The Enzymatic Method

The Antibody Titer In Saline, In Albumin
The Indirect Coombs Tests.

Diagnosis Maternal Isoimmunization

Antibody Titre in Saline: RhD-positive cells suspended in
saline solution are agglutinated by IgM anti-RhD antibody, but not
IgG anti-RhD antibody. Thus, this test measure IgM, or recent
antibody production.

Antibody Titre in Albumin: Reflects the presence of any antiRhD IgM or IgG antibody in the maternal serum.

The Indirect Coombs Test:

o First Step:
RhD-positive RBCs are incubated with maternal serum
Any anti-RhD antibody present will adhere to the RBCs.
o Second Step:
The RBCs are then washed and suspended in serum containing
antihuman globulin (Coombs serum).
Red cells coated with maternal anti-RhD will be agglutinated by
the antihuman globulin (positive indirect Coombs test).

The Direct Coombs Test

Is Done After Birth To Detect The Presence Of Maternal
Antibody On The Neonate's RBCs.
The Infant's RBCs Are Placed In Coombs Serum.
If The Cells Are Agglutinated This Indicate The Presence Of
Maternal Antibody

Interpretation of Maternal Anti-D Titer

Antibody Titer Is A Screening Test.
A Positive Anti-d Titer Means That The Fetus Is At Risk For
Hemolytic Disease, Not That It Has Occurred Or Will
Develop.
Variation In Titer Results Between Laboratories And
Intra Laboratory Is Common.
A Truly Stable Titer Should Not Vary By More Than One
Dilution When Repeated In A Given Laboratory.

Pathogenesis of The HD of the Fetus and Newborn

Fetal Rhesus Determination

RHD Type And Zygosity (If RHD-positive) Of The Father

Amniocentesis To Determine The Fetal Blood Type Using
The Polymerase Chain Reaction (PCR)

Detection Of Free Fetal RHD DNA (FDNA) Sequences In

Maternal Plasma Or Serum Using PCR

Flow Cytometry Of Maternal Blood For Fetal Cells

Pathogenesis of Fetal Hemolytic Disease

Methods of Diagnosis and Evaluation of Fetal Rh

Isoimmunization
Measurements Of Antibodies in Maternal Serum

Determination of Fetal Rh Blood Group

Ultrasonography
Amniocentesis
Fetal Blood Sampling

Ultrasonography:

To Establish The Correct Gestational Age.

In Guiding Invasive Procedures And Monitoring Fetal
Growth And Well-being.

Ultrasonographic Parameters To Determine Fetal Anemia:

o Placental Thickness.
o Umbilical Vein Diameter
o Hepatic Size.
o Splenic Size.
o Polyhydramnios.
o Fetal Hydrops (e.g. Ascites, Pleural Effusions, Skin
Edema).

Doppler Velocimetry Of The Fetal Middle Cerebral

Artery (MCA)
Anemic Fetus Preserves Oxygen Delivery To The
Brain By Increasing Cerebral Flow Of Its Already
Low Viscosity Blood.

For Predicting Fetal Anemia

To Predict The Timing Of A Second Intrauterine
Fetal Transfusion.

Invasive Techniques
( Amniocentesis and Fetal Blood Sampling):
Indications:
A

Critical Anti-D Titer:

I.E. A Titer Associated With A Significant Risk For Fetal

Hydrops. Anti-D Titer Value Between 8 And 32

Previous Seriously Affected Fetus Or Infant

(e.g. Intrauterine Fetal Transfusion, Early Delivery, Fetal
Hydrops, Neonatal Exchange Transfusion).

Amniocentesis
Normally Bilirubin In Amniotic Fluid Decreases With
Advanced Gestation.
It Derives From Fetal Pulmonary And Tracheal Effluents.
Its Level Rises in Correlation With Fetal Hemolysis.
Determination Of Amniotic Fluid Bilirubin:
By The Analysis Of The Change In Optical Density Of
Amniotic Fluid At 450 nm On The Spectral Absorption Curve
(delta OD450)
Procedures Are Undertaken At 10-15 Days Intervals Until
Delivery Data Are Plotted On A Normative Curve Based Upon
Gestational Age.

Extended Liley graph.

Queenan curve (Deviation in amniotic fluid optical density at a wavelength of 450

nm in Rh-immunized pregnancies from 14 to 40 weeks' gestation)

Interpretation Of Amniotic Fluid Bilirubin:

A Falling Curve: Is Reassuring: i.e. An Unaffected Or

RhD-negative Fetus.

A Plateauing Or Rising Curve: Suggests Active

Hemolysis (Require Close Monitoring And May Require
Fetal Blood Sampling And/Or Early Delivery).

A Curve That Reaches To Or Beyond The 80th

Percentile Of Zone II On The Liley Graph Or Enters The
Intrauterine Transfusion" Zone Of The Queenan Curve:
Necessitates Investigation By Fetal Blood Sampling

Fetal blood sampling:

Is the gold standard for detection of fetal anemia.
Reserved for cases with:

- With an increased MCA-PSV

- Increased OD 450

Complications:
Total Risk of Fetal Loss Rate 2.7% (Fetal death is 1.4%
before 28 weeks and The perinatal death rate is 1.4% after
28 weeks).
Bleeding from the puncture site in 23% to 53% of cases.
Bradycardia in 3.1% to 12%.
Fetal-maternal hemorrhage: occur in 65.5% if the placenta
is anterior and 16.6% if the placenta is posterior.
Infection and abruptio placentae are rare complications

MONOCLONAL ANTI-D
Most polyclonal RhIg comes from male volunteers who are intentionally
exposed to RhD-positive red blood cells.
Potential Problems:
infectious risk
solve supply problems.
ethical issues

anti-D monoclonal antibody:

Although monoclonal anti-D is promising, it cannot be recommended at this
time as a replacement for polyclonal RhIg.

Complications of Fetal-Neonatal Anemia:

Fetal Hydrops And Stillbirth
Hepatosplenomegaly
Neonatal Jaundice
Compilations Of Neonatal Kernicterus (Lethargy,
Hypertonicity, Hearing Loss, Cerebral Palsy And
Learning Disability)
Neonatal Anemia

Causes Of Fetal Neonatal Anemia:

Blood Loss:
o Abnormal Placental Separation (Abruptio Placentae) Or Placenta Previa
o Traumatic Tear Of The Umbilical Cord
o Occult Blood Loss In Utero As A Result Of Fetomaternal Hemorrhage.
o A Chronic Twin-to-twin Transfusion In Identical Twins

Alloimmune Hemolytic Disease Of The Newborn (HDN):

Anemia Due To Congenital Spherocytosis
Nonspherocytic Hemolytic Anemias
Infections:
Hemoglobinopathies:

The RH Antigen

Diagnostic algorithm for neonatal anemia. *Note that the direct antiglobulin (Coombs)

Monthly Maternal Indirect Coombs Titre

Exceeds Critical Titre

Below Critical Titre

Paternal Rh Testing
Rh Positive

Rh-negative

Amniocentesis for RhD antigen status

Fetus RhD positive

Routine Care

Fetus RH D Negative
Weekly MCA-PSV

Serial Amniocentesis

> 1.50 MOM

Cordocentesis or Deliver
Suggested management of the RhD-sensitized pregnancy

< 1.50 MOM

Monthly Maternal Indirect Coombs Titre

Exceeds Critical Titre

Below Critical Titre

Paternal Rh Testing
Rh-negative

Rh Positive
Amniocentesis for RhD antigen status

Fetus RhD positive

Serial Amniocentesis

Routine Care

Fetus RH D Negative
Weeklyl MCA-PSV
< 1.50 MOM

>1.5 MOM
Cordocentesis or De

Suggested management of the RhD-sensitized pregnancy

Serial Amniocentesis
Lily zone I
Lower Zone II

Upper Zone II

Repeat
Amniocentesis every
2-4 weeks

Zone III
Hydramnios & Hydrops

< 35 to 36 weeks
And Fetal lung
immaturity

Delivery at or near term

> 35 to 36
weeks Lung
maturity
present

Intrauterine
Transfusion
Repeat Amniocentesis in 7
days or FBS
Hct < 25%
Intrauterine
Transfusion

Delivery

Hct > 25%

Repeat Sampling
7 to 14 days

Suggested management after amniocentesis for OD 450

Serial Amniocentesis
Lily zone I
Lower Zone II

Upper Zone II

Zone III
Hydramnios & Hydrops

Repeat Amniocentesis
every 2-4 weeks
< 35 to 36 weeks
And Fetal lung immaturity

> 35 to 36 weeks
Lung maturity present

Delivery at or near term

Intrauterine
Transfusion

Delivery

Repeat Amniocentesis or FBS

Hct < 25%
Intrauterine
Transfusion

Hct > 25%

Repeat Sampling
7 to 14 days

Suggested management after amniocentesis for OD 450

Average regression lines for healthy fetuses (dotted line), mildly anemic fetuses (thin l
2002;187:938; with permission.)

Suggested management of the patient with antibody screen positive for antigen
other than RhD.

Incidence Of Maternal Alloimmunization

The overall incidence of maternal alloimmunization to clinically significant RBC
antigens has been estimated to be 25 per 10,000 live births

RhD D negativity primarily occurs among Caucasians; the average

incidence is 15 percent in this group. Examples of the blood group
distribution in various populations are illustrated below:
Basques 30 to 35 percent
Finland 10 to 12 percent
American blacks 8 percent
Indo-Eurasians 2 percent
Native Americans and Inuit Eskimos 1 to 2 percent.

Changes since introduction of Anti-D

PATHOGENESIS

Chronic transplacental hemorrhage.

Failure to administer Rh immune globulin when indicated.
or non-detection of a large fetal bleed at delivery

As an example, in a study of 110 pregnant mothers with 111 at-risk

fetuses, and maternal serum titers of 1:16 or greater, antibodies to D,
K, E, and c were present in 84, 18, 8, and 3 fetuses, respectively

The nature of the Rh antigen complex is determined by a specific gene

sequence inherited in a Mendelian fashion from the parents, one haploid from
the mother and one from the father. In 1974 the location of the Rh gene
complex was pinpointed on the distal end of the short arm of chromosome one.
Three genetic loci, each with two possible alleles determined the Rh antigen
(i.e. Rh blood group).

The amount of fetal cells in maternal blood:

the Kleihauer-Braun-Betke test

The severity of fetal anemia is influenced:

Primarily by antibody concentration,
Additional factors that are not fully understood.
These include the subclass and glycosylation of maternal antibodies.
The structure, site density, maturational development and tissue distribution
of blood group antigens.
The efficiency of transplacental IgG transport.
The functional maturity of the fetal spleen.
Polymorphisms which affect Fc receptor function; and the presence of HLArelated inhibitory antibodies [13].

DIAGNOSIS

Blood and Rh(D) typing and an antibody screen should always be performed at the firs

Below the critical titer there is a risk of mild to moderate, but not severe, fetal or
neonatal hemolytic anemia. Fetal assessment with invasive techniques (eg,
amniocentesis, fetal blood sampling) is required when a critical titer is present
or if the patient has had a prior significantly affected pregnancy (eg, intrauterine
fetal transfusion, early delivery, fetal hydrops, neonatal exchange transfusion).
The purpose of these invasive tests is to determine whether severe fetal
anemia is present.

Ultrasonography
A variety of ultrasonographic parameters have been used to determine
whether fetal anemia is present. These parameters include: placental
thickness; umbilical vein diameter; hepatic size; splenic size; and
polyhydramnios

Liver lengths plotted against gestation for 18 fetuses with anemia with
ultrasonographic measurement during week before delivery, shown in
reference to normal values Open circles, Cord hemoglobin level <90
g/L; solid circles, cord hemoglobin level 90 to 130 L.

Liver length measurements made within 48 hours of fetal blood sampling

in all fetuses with anemia at first fetal blood sampling, shown in reference
to normal values.

Ultrasound image of amniocentesis at 16 weeks of gestation

Ultrasound image of transabdominal chorionic villus sampling.

Diagram of cordocentesis procedure

Doppler velocimetry Doppler assessment of the fetal middle cerebral

artery (MCA)

Amniocentesis Amniocentesis is performed when the critical titer is reached

or if there has been a previous seriously affected fetus or infant.

Fetal blood sampling Ultrasound-directed fetal blood sampling (ie,

percutaneous umbilical blood sampling, cordocentesis, funipuncture) allows
direct access to the fetal circulation to obtain important laboratory values such as
hematocrit, direct Coombs, fetal blood type, reticulocyte count, and total bilirubin

Multiple antibodies Some women develop antibodies to more than one red
blood cell antigen.

Ultrasound image of cordocentesis with the needle tip located in a free loop of c

Ultrasound-guided transabdominal fetocentesis

Ultrasound image of bladder outlet obstruction with enlarged bladder, classic

keyhole appearance seen with posterior urethral valves, and anhydramnios

Double pig-tailed Rocket catheter and trocar used for vesicoamniotic shunting.