B l ood B anking
Hemolytic Disease of the
Fetus and Newborn
 Fetal red cells become coated with IgG
alloantibody of maternal origin directed
against a paternally inherited antigen
present on the fetal cells that are absent
from maternal cells.
 The destruction of the red blood cells
(RBCs) of a fetus and neonate by
antibodies produced by the mother.
 95% of the cases of HDFN were caused
by maternal antibodies directed against
the Rh.
 Stimulated to form antibodies by
previous transfusion or pregnancy, and
during the 2nd or 3rd Trimester.
 Cause: antibodies in the mother directed
against the Rh antigen D
 In 1968- introduction of Rh-immune
globulin (RhIG) lead to decrease of the
incidence of the disease.
 Rh(D) incompatibility is common
Advances in the Diagnosis and Treatment
of HDFN
 Ultrasonography,
 Doppler assessment of middle
cerebral
 Artery peak systolic velocity
 Cordocentesis
 Allele-specific gene amplification
studies on fetal cells in amniotic
fluid
 Fetal DNA analysis in maternal
plasma
 Intravascular intrauterine transfusion
By: Elly
Etiology
 17TH Century- clinical findings of
the fetus and newborn were noted
 1939- Levine and Stetson reported a
transfusion reaction from transfusing
a husband’s blood to a postpartum
woman.
o They postulated that- the
mother had been immunized
to the father’s antigen
through fetomaternal
hemorrhage
 Immunoglobulin G (IgG) class-
actively transported across the
placenta; other immunoglobulin
classes, such as IgA and IgM, are
not.
o Most IgG antibodies are
directed against bacterial,
fungal, and viral antigens,
so the transfer of IgG from
the mother to the fetus is
beneficial
o In HDFN- the antibodies are
directed against those
antigens on the fetal RBCs
that were inherited from the
father.
Rh HDFN
 Rh-positive firstborn infant of a
Rh-negative mother usually is
unaffected because the mother has
not yet been immunized.
 During gestation and delivery- the
placenta separates from the uterus,
variable numbers of fetal RBCs enter
the maternal villus sampling and
trauma to the abdomen can increase
the risk of fetomaternal
hemorrhage.
 B l ood B anking By: Elly

 Volume of fetomaternal hemorrhage:

Small, however, as little as 1 mL of
Antibody Specificity
fetal RBCs can immunize the
mother
The number of antigens on the fetal RBCs
corresponds to heterozygous RBCs,
because all fetal antigens incompatible with
the mother have been inherited from the
father, who can give only one set of genes
to the fetus.
Host Factors
 The ability of individuals to
produce antibodies in response to
antigenic exposure varies, depending
on complex genetic factors
 In Rh-negative individuals who are
transfused with 200 mL of Rh-
positive RBCs, approximately 85%
respond and form anti-D
 If RhIG is not administered, the
risk of immunization is only about
16% for a Rh-negative mother
after a Rh-positive pregnancy.
 Among the RBC antigens, D is the
most antigenic
 Only Rh-negative blood is
transfused to Rh-negative females
of childbearing potential
 Potent immunogens but are less
than D
- C, E and c (associated with
moderate to severe cases of

HDFN)
Anti-Kell- non–Rh system
antibodies, considered the most
clinically significant in its ability to
cause HDFN
o Kell antigens are present on
immature erythroid cells in
the bone marrow, so severe
anemia occurs not only by
destruction of circulating
RBCs but also by precursors
 All pregnant women with IgG RBC
antibodies should be followed
closely for HDFN

Antibodies Identified in Prenatal Specimens as a

Immunoglobulin Class Cause of HDFN. According to Vengelen-Tyler
COMMON RARE NEVER
 Affects the severity of HDFN Anti-D Anti-Fya Anti-Lea
 ONLY IgG can cross placenta Anti-D + C Anti-s Anti-Leb
 Active transport of IgG begins in the Anti-D + E Anti-M Anti-I
second trimester and continues until Anti-C Anti-N Anti-IH
birth. Transported via the FC Anti-E Anti-S Anti-P1
region Anti-c Anti-Jka
 IgG1 and IgG3- more efficient in Anti-e
hemolysis Anti-K
 All subclasses of IgG are
transported across the placenta and
can affect the severity of disease. Influence of ABO Group
 If the mother is compatible with the
fetus the incidence of detectable
 B l ood B anking By: Elly

fetomaternal hemorrhage is  May present with mild anemia or normal

decreased. hemoglobin levels, hyperbilirubinemia,
 Fetal RBC are less developed at birth and jaundice within 12-24 hours after
that is why there is a decrease birth.
reaction.
 The ABO incompatibility protects HDFN Caused by RBC
somewhat against Rh immunization,
apparently by the hemolysis of ABO- Alloimmunization
incompatible D-positive fetal RBCs
in the mother’s circulation before the  Fetomaternal Hemorrhage
D antigen can be recognized by her o Transplacental hemorrhage of
immune system fetal RBCs into the maternal
circulation
Pathogenesis is the effect of the maternal  Maternal Factors
antibody on the fetus, the reasons for the o Ability of individuals to
effects, and the ensuing signs and produce in response to
symptoms. antigenic exposure varies
Comparison of ABO Versus RhD HDFN  RBC Antibody Specificity
Characteristic ABO RhD o RhD- most antigenic
First pregnancy can Yes Rare o Anti-Kell- most significant
be affected non-Rh Ab that causes
Disease predicted by No Yes HDFN
titers
Causative antibody Yes Yes (anti-D, Hemorrhage of D-positive fetal RBCs into
IgG (anti-A, etc) D-negative mother
B)
Bilirubin level at Normal Elevated Maternal antibody formed against
birth range paternally inherited D antigen
Intrauterine None Sometimes
transfusion needed Next-D positive pregnancy
Anemia at birth No Yes
Phototherapy Yes Yes Placental passage of maternal IgG anti-D
beneficial
Exchange transfusion Rare Uncommon
Maternal antibody attaches to fetal RBCS
needed
Hemolysis of fetal RBCs

 Most common cause but less severe

 IgG Ab almost always occurs in the
mother’s circulation without a history of
prior exposure
 Can occur in any pregnancy
 Usually among group A or B infants
born to group O mothers
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Pathophysiology
RBC Destruction
 Hemolysis- Maternal IgG attaches to
specific antigens of the fetal RBCs
o The antibody coated cells are
removed from the circulation
by the macrophages of the
spleen
o The rate of RBC
destruction depends on
antibody titer and
specificity and on the number
of antigenic sites on the fetal
RBCs
o Destruction of fetal RBCs
and the resulting anemia
stimulate the fetal bone
marrow to produce RBCs at
an accelerated rate, even to
the point that immature RBCs
(erythroblasts) are released
into the circulation
 Erythropoiesis- increased
destruction stimulates the fetal bone
marrow to produce more RBCs
(immature)
By: Elly
 Anemia- increased RBC destruction
Hydrops Fetalis
 Condition wherein there is
abnormal interstitial fluid
collection in two or more
compartments of the fetal body
 Severe anemia and
hypoproteinemia (caused by
decreased hepatic production of
plasma proteins) lead to the
development of high-output cardiac
failure with generalized edema,
effusions, and ascites, a condition
known as hydrops fetalis
 Severe cases: develop by 18 to 20
weeks’ gestation.
Two types
1. Immune- associated with red cell
alloimmunization
o Erythroblastosis fetalis- results
from antibodies in the maternal
circulation that reacts with fetal
antigens resulting in fetal
hemolysis. When the bone
marrow fails to produce enough
RBCs to keep up with the rate of
RBC destruction, erythropoiesis
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outside the bone marrow is
increased in the hematopoietic
tissues of the spleen and liver
o These organs become enlarged
(hepatosplenomegaly), resulting
in portal hypertension and
hepatocellular damage.
2. Non-immune
 The process of RBC destruction
continues even after such an infant is
delivered alive. continues as long as
maternal antibody persists in the
newborn infant’s circulation.
 The rate of RBC destruction after
birth decreases because no additional
maternal antibody is entering the
infant’s circulation through the
placenta.
 IgG is distributed both
extravascularly and intravascularly
and has a half-life of 25 days, so
antibody binding and hemolysis of
RBCs continue for several days to
weeks after delivery.
Bilirubin
Pathophysiology: Icterus
Gravis Neonatorum
By: Elly
 Physiologic jaundice of the newborn
Vs. HDFN
 RBC destruction releases
hemoglobin, which is metabolized to
Indirect bilirubin. The indirect
bilirubin is transported across the
placenta and conjugated in the
maternal liver to “direct” bilirubin.
The conjugated bilirubin is then
excreted by the mother.
 Levels of total bilirubin in the fetal
circulation and amniotic fluid may be
elevated, after birth, accumulation of
metabolic by-products of RBC
destruction can become a severe
problem
 The newborn liver is unable to
conjugate bilirubin efficiently,
especially in premature infants.
With moderate to severe hemolysis,
the unconjugated, or indirect,
bilirubin can reach levels toxic to the
infant’s brain.
o more than 18 to 20 mg/dL
 Marked hyperbilirubinemia
 Kernicterus or permanent damage
to parts of the brain – bilirubin
deposition in the brain
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Diagnosis and
Management
Serologic and clinical tests performed at
appropriate times during the pregnancy can
accurately determine the level of antibody
in the maternal circulation, the potential of
the antibody to cause HDFN, and the
severity of RBC destruction during gestation
Serologic Testing of the
Mother
Recommended: perform the type and
antibody screen at the first prenatal visit,
preferably during the first trimester.
 Post-Natal Diagnosis
- DAT on cord or neonatal RBCs is
the most significant diagnostic test
- DAT (+) helps confirm HDFN
 ABO, RhD and Antibody Screen
- Pre-natal specimen typed for ABO
and RhD.
- Perform IAT
- The antibody screening method
must be able to detect clinically
significant IgG alloantibodies that
are reactive at 37°C and in the
antiglobulin phase
- Two separate reagent screening
cells, covering all common blood
group antigens (preferably
homozygous), should be used.
By: Elly
- Tube testing: use enhancing medium
(PEG, LISS,)
- Prenatal patients may produce
clinically insignificant antibodies,
such as anti-Lea or anti-Leb,
immediate spin and room temperature
are omitted to reduce detection of
IgM that cannot cross placenta.
- Solid phase or gel column may be
used
- If the antibody screen is
nonreactive, repeat testing is
recommended before RhIG therapy in
Rh-negative prenatal patients and in
the third trimester if the patient has
been transfused or has a history of
unexpected antibodies
 Antibody Identification
- If the antibody screen is reactive
- Follow-up testing will depend on the
antibody specificity.
- Cold reactive IgM antibodies such
as anti-I, anti-IH, anti-Lea, anti-Leb,
and anti-P1 can be ignored
- Anti-M and anti-N can cause mild to
moderate HDFN
- To establish the immunoglobulin
class, the serum can be treated with a
sulfhydryl reagent, such as
dithiothreitol or 2-
mercaptoethanol, and then retested
with appropriate controls to destroy
the J chain of IgM and IgG will
remain active.
- Weakly reactive anti-D- during 3rd
trimester
- Receive RhIG- either after an event
with increased risk of fetomaternal
hemorrhage or at 28 weeks’ gestation
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(antenatal), passively administered
anti-D will be weakly reactive in
testing and will remain demonstrable
for 2 months or longer.
- Active immunization: titer higher
than 4
- Titer under 4- Immunization cannot
be ruled out.
- Other than anti-D, the most common
and most significant antibodies are
anti-K, anti-E, anti-c, anti-C, and
anti-Fya
Management of the
Fetus
 Fetal Ultrasound
- Middle Cerebral Artery Peak Systolic
Velocity (MCA-PSV) with color
Doppler ultrasonography
o At about 16 to 20 weeks’
gestation, further diagnosis
and treatment are begun.
o Patients with a history of a
severely affected fetus or
early fetal death may
require earlier intervention.
o Predict anemia in fetus
o Color Doppler indicates the
direction of blood flow, using
red for arterial flow and blue
for venous
o The middle cerebral artery
is used because of its easy
accessibility.
By: Elly
o The measurement is based
on the reduced blood
viscosity at low hematocrit
and the resulting faster
velocity.
o Noninvasive and poses no
adverse effects for the fetus.
 Intrauterine Transfusion
- Invasive procedure of transfusing
blood via umbilical cord.
- Performed by cordocentesis and
injecting donor RBCs directly into the
vein
- Suppresses the fetal bone marrow
RBC production. During the first
weeks after birth, the infant may
require additional RBC
transfusion.
- Goal: maintain fetal hemoglobin
above 10 g/dL
- Procedure is repeated every 2 to 4
weeks until delivery
- The initial intrauterine transfusion
is rarely performed after 36 weeks’
gestation.
- Necessary when one or more of the
following conditions exists:
 MCA-PSV indicates anemia
 Fetal hydrops is noted on
ultrasound examination
 Cordocentesis blood sample
has hemoglobin level less than
10 g/dL.
 Amniotic fluid ΔOD 450 nm
results are high.
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Note: Cordocentesis, intrauterine
transfusion, and amniocentesis have
several risks, including infection, premature
labor, and trauma to the placenta, which may
cause increased antibody titers
 Cordocentesis
- Invasive procedure of collecting cord
blood for testing
- Using high-resolution ultrasound
with color Doppler enhancement of
blood flow, the umbilical vein is
visualized at the level of the cord
insertion into the placenta
- A spinal needle is inserted into the
umbilical vein, and a sample of the
fetal blood is obtained.
- The fetal blood sample can then be
tested for hemoglobin, hematocrit,
bilirubin, blood type, direct
antiglobulin test (DAT), and
antigen phenotype and genotype.
 Amniocentesis
- Invasive procedure of collecting
amniotic fluid for testing
By: Elly
- Uncommon, and MCA-PSV gives
the same information.
- The concentration of bilirubin
pigment in the amniotic fluid
estimates the extent of fetal
hemolysis
- Tested by a spectrophotometric scan
at steadily increasing wavelengths,
so the change in the optical density
(ΔOD) at 450 nm
- An increasing or unchanging ΔOD
450 nm as pregnancy proceeds
predicts worsening of the fetal
hemolytic disease
- Fetal hemoglobin less than 8 g/dL-
indicate severe and often life-
threatening hemolysis
Management of the Infant
 Phototherapy
- Used to metabolize unconjugated
bilirubin
 Intravenous Immune Globulin (IVIG)
- Competes with the mother’s
antibodies for the FC receptors on the
macrophages in the infant’s spleen.
Assessment of
Fetomaternal Hemorrhage:
ROSETTE TEST
(Qualitative Test)
 Screening test for FMH
 Detects fetal D positive red cells in
maternal Rh-negative blood
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 (+): formation of small clumps of RBC
 If positive, proceed to KBT or FC

KLEIHAUR-BETKE
Key Points
TEST (Quantitative Test)
 Maternal blood smear is treated with
acid and then stained with a
counterstain
 Fetal cells- stained pink
 Maternal cells- unstained (ghost
cells)
Number of fetal cells X Maternal blood volume
Number of maternal cells
Volume of fetomaternal hemorrhage
Rhogam Dosage
 The following calculations should be
used to determine an additional
number of vials of Rho(D) immune
globulin needed if the KB test is
positive, indicating a large amount of
FMH. One vial contains 300 mcg
and will protect against 30 ml of fetal
blood
 Volume (ml) of fetal blood=
Percentage of fetal cells X 50
 Number of Vials of 300 mcg RhUG
required= Volume of fetal
blood/30ml
 If the number to the right of the
decimal point is less than 5, round
down and add one vial
By: Elly
 If the number to the right of the
decimal point is greater than or equal
to 5, round up and add one vial.
 In ABO HDFN, the firstborn infant
may be affected as well as
subsequent pregnancies in which the
mother is group O and the newborn
is group A, B, or AB; the IgG
antibody, anti-A,B in the mother’s
circulation, crosses the placenta and
attaches to the ABO-incompatible
antigens of the fetal RBCs.
 Erythroblastosis fetalis describes the
presence of immature RBCs or
erythroblasts in the fetal circulation
because the splenic removal of the
IgG-coated RBCs causes anemia; the
term commonly used now is HDFN
 Prenatal serologic tests for obstetric
patients include an ABO, Rh, and
antibody screen during the first
trimester of pregnancy.
 Cord blood workup includes tests for
ABO and Rh as well as DAT; the
most important serologic test for
diagnosis of HDFN is the DAT with
anti-IgG reagent.
 RhIG administered to the mother
within 72 hours following delivery is
used to prevent active immunization
by the Rh(D) antigen on fetal cells;
RhIG attaches to fetal Rh-positive
RBCs in maternal circulation,
 B l ood B anking By: Elly

blocking immunization and

subsequent production of anti-D.

 A Kleihauer-Betke test or flow

cytometry is used to quantitate the
number of fetal Rh-positive cells in
the mother’s circulation as a result of
a fetomaternal hemorrhage.