Rh Isoimmunization

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Rh Isoimmunization
Professor Hassan A Nasrat Chairman of the
Department of Obstetrics and Gynecology Faculty
of Medicine King Abdul-Aziz University
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ISO is a prefix means similar, equal or
uniform. Isoimmunization is the process of
immunizing a species with antigen derived from
the same subject.
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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.
4
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)

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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
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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.
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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.

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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.
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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.
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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.

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Phenotype
Genotype
eCd/EcD
D positive

• Antigenicity of the Rh surface protein
• genetic expression of the D allele.
• Number of specific Rh antigen sites.
• Interaction of components of the Rh gene complex.
• Exposure of the D antigen on the surface of the
  red cell

e
C
d
D
c
E
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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).
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• 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.

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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.
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Natural History of Rh Isoimmunization And HD
Fetus and Newborn

• 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.

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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

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• 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

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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.

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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
• First Step
• RhD-positive RBCs are incubated with maternal
  serum
• Any anti-RhD antibody present will adhere to the
  RBCs.
• 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).

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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
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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.

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Pathogenesis of The HD of the Fetus and Newborn
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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

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Pathogenesis of Fetal Hemolytic Disease
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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

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Ultrasonography

• To Establish The Correct Gestational Age.
• In Guiding Invasive Procedures And Monitoring
  Fetal Growth And Well-being.
• Ultrasonographic Parameters To Determine Fetal
  Anemia
• Placental Thickness.
• Umbilical Vein Diameter
• Hepatic Size.
• Splenic Size.
• Polyhydramnios.
• Fetal Hydrops (e.g. Ascites, Pleural Effusions,
  Skin Edema).

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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.

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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).

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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.
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Extended Liley graph.
31
Queenan curve (Deviation in amniotic fluid
optical density at a wavelength of 450 nm in
Rh-immunized pregnancies from 14 to 40 weeks'
gestation)
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• 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

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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

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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.
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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

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Causes Of Fetal Neonatal Anemia
Blood Loss

• Abnormal Placental Separation (Abruptio
  Placentae) Or Placenta Previa
• Traumatic Tear Of The Umbilical Cord
• Occult Blood Loss In Utero As A Result Of
  Fetomaternal Hemorrhage.
• 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
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The RH Antigen
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Diagnostic algorithm for neonatal anemia. Note
that the direct antiglobulin (Coombs) test can be
negative or weakly positive despite the presence
of ABO incompatibility and hemolysis. (Adapted
from Blanchette VS, Zipursky A. Assessment of
anemia in newborn infants. Clin Perinatol
198411(2)489510 with permission.)
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Monthly Maternal Indirect Coombs Titre
Below Critical Titre
Exceeds Critical Titre
Paternal Rh Testing
Rh Positive
Rh-negative
Amniocentesis for RhD antigen status
Routine Care
Fetus RhD positive
Fetus RH D Negative
Weekly MCA-PSV
Serial Amniocentesis
lt 1.50 MOM
gt 1.50 MOM
Cordocentesis or Deliver
 Suggested management of the RhD-sensitized
pregnancy
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Monthly Maternal Indirect Coombs Titre
Below Critical Titre
Exceeds Critical Titre
Paternal Rh Testing
Rh-negative
Rh Positive
Amniocentesis for RhD antigen status
Routine Care
Fetus RhD positive
Fetus RH D Negative
Weeklyl MCA-PSV
Serial Amniocentesis
lt 1.50 MOM gt1.5 MOM
Cordocentesis or Deliver
 Suggested management of the RhD-sensitized
pregnancy
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Serial Amniocentesis
Lily zone I Lower Zone II
Zone III Hydramnios Hydrops
Upper Zone II
Repeat Amniocentesis every 2-4 weeks
lt 35 to 36 weeks And Fetal lung immaturity
gt 35 to 36 weeks Lung maturity present
Delivery at or near term
Intrauterine Transfusion
Repeat Amniocentesis in 7 days or FBS
Delivery
Hct lt 25
Hct gt 25
Intrauterine Transfusion
Repeat Sampling 7 to 14 days
Suggested management after amniocentesis for ?OD
450
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Serial Amniocentesis
Lily zone I Lower Zone II
Upper Zone II
Zone III Hydramnios Hydrops
Repeat Amniocentesis every 2-4 weeks
lt 35 to 36 weeks And Fetal lung immaturity
gt 35 to 36 weeks Lung maturity present
Delivery at or near term
Intrauterine Transfusion
Delivery
Repeat Amniocentesis or FBS
Hct lt 25
Hct gt 25
Intrauterine Transfusion
Repeat Sampling 7 to 14 days
Suggested management after amniocentesis for ?OD
450
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Average regression lines for healthy fetuses
(dotted line), mildly anemic fetuses (thin line),
and severely anemic fetuses (thick line). (From
Detti L, Mari G, Akiyama M, Cosmi E, Moise Jr KJ,
Stefor T, et al. Longitudinal assessment of the
middle cerebral artery peak systolic velocity in
healthy fetuses and in fetuses at risk for
anemia. Am J Obstet Gynecol 2002187938 with
permission.)
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Suggested management of the patient with antibody
screen positive for antigen other than RhD.
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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
49
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   America
n blacks 8 percent   Indo-Eurasians 2
percent   Native Americans and Inuit Eskimos
1 to 2 percent.
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Changes since introduction of Anti-D
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PATHOGENESIS

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

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As an example, in a study of 110 pregnant mothers
with 111 at-risk fetuses, and maternal serum
titers of 116 or greater, antibodies to D, K, E,
and c were present in 84, 18, 8, and 3 fetuses,
respectively
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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).
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The amount of fetal cells in maternal blood
the Kleihauer-Braun-Betke test
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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.
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DIAGNOSIS
Blood and Rh(D) typing and an antibody screen
should always be performed at the first prenatal
visit
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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.
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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
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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 lt90 g/L solid circles, cord
hemoglobin level 90 to 130 L.
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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.
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Ultrasound image of amniocentesis at 16 weeks of
gestation
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Ultrasound image of transabdominal chorionic
villus sampling.
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Diagram of cordocentesis procedure
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Doppler velocimetry Doppler assessment of the
fetal middle cerebral artery (MCA)
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  Amniocentesis Amniocentesis is performed when
the critical titer is reached or if there has
been a previous seriously affected fetus or
infant.
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  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
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Multiple antibodies Some women develop antibodies
to more than one red blood cell antigen.
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Ultrasound image of cordocentesis with the needle
tip located in a free loop of cord.
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Ultrasound-guided transabdominal fetocentesis
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Ultrasound image of bladder outlet obstruction
with enlarged bladder, classic keyhole appearance
seen with posterior urethral valves, and
anhydramnios
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Double pig-tailed Rocket catheter and trocar used
for vesicoamniotic shunting.
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