Alloimmune Hemolytic Disease Of The Fetus / Newborn:

1
Alloimmune Hemolytic Disease Of The Fetus /
Newborn 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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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. The mother become
Isoimmunized. e.g. When An RhD Negative Mother
Carries An RhD Positive Fetus and Develop
Isoimmunistion. In The Fetus Erythroblastosis
Fetalis In The Newborn HDN.
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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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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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• 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.
• Local Studies (population) 8

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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
• Pathogenesis of Fetal Erythroblastosis Fetalis
• Diagnosis of Rh isoimmunization

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The RH Antigen Biochemical and Genetic Aspects
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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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Mechanism of Development of Maternal Rh
Isoimmunization
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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 Explains the development of
fetal isoimmunization in a primigravida, who has
no history of exposure to incompatible Rh blood.
Rh negative Fetus and the mother is Rh
positive The Fetus is exposed to the maternal
Rh antigen through maternal-fetal transplacental
bleed. 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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Mother
1. Cleared by Macrophage
Primary Response
2. Plasma stem cells

• 6 wks to 6 M.
• IGM.

IGM antibodies
Placental
Fetal Anaemia
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Mother
Macroph. antigen Presenting cell
T- helper cell
Secondary Response

• Small amount
• Rapid
• IgG

B cell
IgG
Anti - D
Placental
Fetal Anaemia
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Mother
Macroph. Antigen Presenting Cell
Group O Rh Negative
T-Hellper
Anti - A
Anti - B
B-cell
Anti-D
Placenta
A Rh positive
B Rh Positive
Infant
O Rh positive
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Natural History of Maternal isoimmunization /HD
of the Newborn
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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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Pathogenesis of Fetal Erythroblastosis Fetalis
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Rh Antibodies Antibodies Coated Red
Cells Destruction of Fetal Cells by Fetal
RES Fetal Anemia Fetal Hypoxia and Stimulate of
Erythropoitin Extra Medullary red Cells
Synthesis Hepatomegally Hepatic Cell
Failure Hypoproteinemia, Increased Intrahepatic
Pressure, Portal hypertension Ascetic, Edema,
hypoxia, Placental Thickness, Polyhydramnios,
Pericardial effusion
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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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Management
- Prevention
- Treatment of Diagnoses cases of Maternal
Isoimmunisation
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Prevention of Rh Isoimmunization
Screening all women for D Factor and antibodies
Prophylaxis (Anti D Immunoglobulin) only for
those who are negative for antibodies
Anti D Is given 72 hours after delivery, 28-32
weeks, and any other time when there is risk of
Fetomateranl Bleeding
The dose of Immunoglobulin depends the volume of
Blood
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Dose of prophylactic Anti-D Ig
10 mcg of anti-D Ig should be administered for
every mL of fetal blood in the maternal
circulation. Thus, the 300-mcg dose is more
than adequate for a typical feto- maternal hem-
orrhage and covers hemorrhage volumes up to 30 mL
of whole fetal blood. In the less than 1 of
cases where the volume of fetomaternal hemorrhage
exceeds 30 mL, utilizing the Kleihauer-Betke test
to quantitate the volume of fetomaternal hemo-
rrhage and admini- stering the appropriate amount
of anti-D Ig (10 mcg/mL fetal blood) is necessary
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The amount of fetal cells in maternal blood
The Kleihauer-Braun-Betke Test
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Management of cases of Rh isoimmunisation

• Diagnosis Of RH Isoimmunisation
• Evaluation of Fetal Condition

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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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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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Management of cases of Rh isoimmunisation

• Diagnosis Of RH Isoimmunisation
• Evaluation of Fetal Condition

38
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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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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Ultrasound Image of Transabdominal Chorion Villus
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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Ultrasound image of amniocentesis at 16 weeks of
gestation
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Extended Liley graph.
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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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Cordocentesis
Diagram of cordocentesis procedure
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Cordocentesis
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Monthly Maternal Indirect Coombs Titre
Below Critical Titre
Complicated History and / or 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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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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Management of cases of Rh isoimmunisation
The goals in managing the alloimmunized
pregnancy are 2-fold Initially detecting
fetal anemia prior to the occurrence of fetal
compromise. Minimize fetal morbidity and
mortality by correcting this anemia until fetal
lung maturity and delivery can be achieved.
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Ultrasound-guided transabdominal fetocentesis
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Past Obstetric History
Although not reliably accurate in predicting
severity of fetal disease, past obstetrical
history can be somewhat prognostic
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Antibody Titer in maternal blood
Titers greater than 14 should be considered Rh
alloimmunized. However, the threshold for
invasive fetal testing varies at different
institutions and generally is 116 or greater
because these titers have been associated with
fetal hydrops
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spectrophotometric measurements of bilirubin in
amniotic fluid
Because the wavelength at which bilirubin
absorbs light is 420-460 nm, the amount of shift
in optical density from linearity at 450 nm (D OD
450) in serial amniotic fluid samples can be used
to estimate the degree of fetal
hemolysis. Modification of the Liley curve to
adjust for the relative inaccuracy of D OD 450
readings in early-to-middle second trimester and
the use of serial measurements has improved its
accuracy.
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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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Changes Since Introduction of Anti-D
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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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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.