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:
genetic expression of the D
eCd 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 anti-

RhD 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 ΔOD 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 Routine Care

Fetus RhD positive Fetus RH D Negative

Serial Amniocentesis Weekly MCA-PSV

> 1.50 MOM < 1.50 MOM

Cordocentesis or Deliver

Suggested management of the RhD-sensitized pregnancy

 Monthly Maternal Indirect Coombs Titre

Exceeds Critical Titre Below Critical Titre

Paternal Rh Testing

Rh Positive Rh-negative

Amniocentesis for RhD antigen status Routine Care

Fetus RhD positive Fetus RH D Negative

Serial Amniocentesis Weeklyl MCA-PSV

< 1.50 MOM >1.5 MOM

Cordocentesis or De
Suggested management of the RhD-sensitized pregnancy
 Serial Amniocentesis

Lily zone I Upper Zone II Zone III

Lower Zone II Hydramnios & Hydrops

Repeat
Amniocentesis every < 35 to 36 weeks > 35 to 36
2-4 weeks And Fetal lung weeks Lung
immaturity maturity
Delivery at or near term present
Intrauterine
Transfusion

Repeat Amniocentesis in 7 Delivery

days or FBS

Hct < 25% Hct > 25%

Intrauterine Repeat Sampling

Transfusion 7 to 14 days
Suggested management after amniocentesis for ΔOD 450
 Serial Amniocentesis

Lily zone I Upper Zone II Zone III

Lower Zone II Hydramnios & Hydrops

Repeat Amniocentesis
every 2-4 weeks
< 35 to 36 weeks > 35 to 36 weeks
And Fetal lung immaturity Lung maturity present
Delivery at or near term
Intrauterine Delivery
Transfusion

Repeat Amniocentesis or FBS

Hct < 25% Hct > 25%

Intrauterine Repeat Sampling

Transfusion 7 to 14 days

Suggested management after amniocentesis for ΔOD 450

Average regression lines for healthy fetuses (dotted line), mildly anemic fetuses (thin l
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 HLA-
related 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.