CHAPTER TEN

Hemolytic Disease of
the Fetus and New
born (HDFN)
Objectives
 Define the term hemolytic disease of the fetus and newborn (HDN)

 Explain a brief history of HDFN

 Understand the mechanism of sensitization

 Understand and describe the most common clinical features of

HDFN

 Explain the common clinical and laboratory tests required

 Describe the treatments of HDFN

 Understand and describe the prevention of HDFN

Outline of the unit
 The definition of hemolytic disease of the fetus and newborn
(HDFN)

 A brief history of HDFN

 The mechanism of sensitization

 Investigations at prenatal and post natal stages

 The clinical features of HDFN

 The treatment of infants suffering from HDFN

 The prevention measures

 Hemolytic anemia
Definition
Hemolytic anemia:
Those anemias which result from increased rate of
red cell destruction in the circulation.

Immune HA:
One of acquired hemolytic anemias resulted from
binding of antibody, complement or antibody and
complement to red cells.
 Definition…..
 In IgM Hemolytic Anemia, complement fixation: IV
hemolysis

 In IgG complement activation is uncommon where red cell

antigens are widely separated

 Hemolysis is typically EXTRAVASCULAR in RES

 In high titer antibody, mononuclear phagocytic cells

destroy red cells in spleen and liver

 Significant rise in UNCONJUGATED and TOTAL bilirubin in

the blood
 Introduction
HDFN is a disease that starts in the utero and causes jaundice,
anemia, and hepatospleenomegali of the infant

 Lifespan of fetal or infants red cells is shortened by the action of

specific antibodies

 The cause of the disease is blood group incompatibility between

the mother and the fetus

 Antibodies derived from the MOTHER and transferred via

placenta
 Reacts with antigen and causes increased destruction of fetal
RBCs
Intro…..
Intro…..
 The severity of disease ranges from mild anemia, to
mental retardation, brain damage, kernicterus, or still birth.

 The severity of the disease depends on:

▪ The number of fetal red cells destroyed
▪ The antibody titer in the maternal circulation
▪ Ability of the fetus to compensate anemia

 ABO antibodies as IgG in some group O individuals and Rh

antibodies are the common causes

 Other blood group antibodies rarely cause are anti-K, anti-

E and anti-C.
Intro…..
 Originally HDN is known as erythroblastosis
fetalis.

 Initially the disease observed in babies from D-

negative women with D-positive mates.

 Initial pregnancies were not usually affected.

 Infants from subsequent pregnancies were often

still born or severely anemic and jaundiced.
Intro…..

Rh immune globulin (RhIG)

 Protects D- negative mothers against the
production of anti- D following delivery.

 Anti-C, anti-c, anti-E, anti-e are not

protected by RhIG and can cause HDN.
Overview of HDFN
 HDN is a condition in which the red blood cells of
a fetus or neonate are destroyed by
Immunoglobulin G (IgG) antibodies produced by
the mother.

 Rh incompatibility is the common cause but ABO

group incompatibility also can cause HDN.

 To occur HDN the mother should be Rh negative,

but the father and the fetus should be Rh
positive.
 Overview…..
Factors that must be present for HDFN to occur:

 The mother must lack the Ag, and, following

exposure to the Ag should produce Abs of the
IgG class

 The Fetus must possess the Antigens

 The Antigen must be well developed at birth

 Overview…..
The actual antibody production depends on;
Genetic make up:
 Mother should lack the trait where fetus possess the trait

Immunnogenicity:
 Antigens vary in numbers of receptor sites
 Vary their immunogenicity
 D, c, and Kell are know to be very potent in stimulation

Transplacental hemorrhage:
 The time of hemorrhage
 The volume of hemorrhage
 Physical trauma like abortion, cordocentesis, injury, ectopic
pregnancy….
Etiology of HDFN
 The placental barrier limits the number of fetal RBCs
entering the maternal circulation during pregnancy and
 thus reduces the chances of Ab production during
pregnancy.

 The first Rh incompatible infant is usually unaffected.

 The number of RBCs is small and insufficient to cause

antibody production.

 In addition high steroid levels and other factors

associated with pregnancy.
 Etiology ........
 At the time of delivery , a fetal RBCs escape into the
maternal circulation through transplacental hemorrhage or
known as feto-maternal hemorrhage (FMH).

 Immunization can result from fetal RBC exposure following:

▪ Abortion/Still birth
▪ Ectopic pregnancy, or
▪ invasive prenatal diagnosis e.g. amniocentesis,
sampling
▪ ante-partum haemorrhage
▪ Abdominal trauma.
Etiology ........
 Ags on the fetal RBC can stimulate the maternal
immune system which results in the production of
IgG antibodies .

 After 6 mns of delivery 7-10% of mothers will

produce detectable amount of antibodies.

 The number of RBCs in the maternal circulation is

related to the chance of antibody production.

 In a subsequent pregnancy the IgG antibodies cross

the placental barrier by active transport
Etiology ........
 IgG is formed as four classes as IgG1, IgG2, IgG3,
and IgG4 antibody classes with varying degree of
ability to cross placenta.

 IgG1 and IgG3 classes possess the greater degree

of destruction

 Hence they are potentially harmful or dangerous

 The antibodies bind to the fetal antigens which

results in RBC destruction by macrophages in
fetal liver and spleen
Etiology ........
 Hemoglobin liberated from the damaged RBCs
metabolized to indirect bilirubin.

 Bilirubin is harmlessly excreted by the mother

 Fetal liver and spleen enlarge as erythropoiesis

increases in an effort to compensate for the
RBC destruction
 Hepato-spleenomegaly
Etiology ........
 If the RBC destruction continues, the fetus
becomes increasingly anemic.

 Erythroblasts are released into the fetal

circulation
 Erythroblastosis Fetalis

 If this condition is left untreated, cardiac failure

can occur accompanied by hydrops fetalis, or
edema and fluid accumulation in fetal peritoneal
and pleural cavities.
Etiology ........

 Thus the greatest threat to the fetus is cardiac

failure resulting from uncompensated anemia.

 Following delivery, red blood cell destruction

continues with the release of indirect bilirubin.

 The new born liver is deficient in glucuronyl

transferase.
Etiology ........

 The indirect bilirubin binds to tissues results

in jaundice.

 Bind with tissues of the CNS and cause

permanent damage (kernicterus)

 Resulting in deafness, mental retardation, or

death.
 Increased fetal red cell destruction

↑↑ unconjugated
SEVERE ANAEMIA bilirubin

Increased Tissue hypoxia, Toxic to CNS,

cardiovascular failure, brain damage
erythropoiesis - Death (Kernicterus)

Hepatosplenomegaly
Erythroblastosis Erythropoiesis in
fetalis liver and spleen
↓↓ Liver albumin synthesis
Plasma Osmotic pressure –tissue
Fig: Pathophysiology of Hemolytic edema
disease of the fetus and the newborn
Placental antibody transfer
 Mother to fetus transfer takes place through placenta

 IgG has placental transfer molecules on Fc portion

 Placental trophoblasts have IgG Fc receptors on

membrane

 IgG bound and transported by Fc receptor across

placenta

 FcRn transports IgG in acidic vesicles or pH<6.5

Transfer…..

 Transfer of antibody is by active process: only

from mother to fetus not in inverse

 In the first 12 weeks, only small amounts of IgG

transferred

 Before the introduction of immunosuppression

therapy with anti-D Ig,
anti-D is the commonest cause of death from
hemolytic disease
Transfer…..
Transfer…..
Classification of HDN

 HDN is often classified into three categories

based on Antibody specificity:

 Rh specific antibodies

 ABO antibodies and

 Other (non-Rh) antibodies.

HDN due to RH incompatibility

 Anti- D is responsible for the most severe

cases of HDN

 In most cases, D-negative women become

alloimmunized (produce anti- D) after the
first D + positive pregnancy

 In rare cases, alloimmunization occurs during

the first pregnancy but rarely results in
clinical signs of HDN
 RH incompatibility ……
 In some cases, the maternal anti-D binds to fetal D+positive
red blood cells and causes a positive direct antiglobulin test
(DAT)
 Moderately affected infants develop signs of

 Jaundice, and
 Corresponding elevations in bilirubin levels, during the
first few days of life.

 Severely affected D- positive infants

 Experience anemia in utero and develop jaundice within
hours of delivery.
Development of HDN

Fig.6-1 Development and prevention of HDN

HDN due to ABO inconpatibility

 Occurs most frequently in group A or B

babies born to group O mothers.
 Is due to the increased incidence of IgG ABO
Abs in group O individuals compared to
other ABO groups.
 ABO incompatibility often affects the first
pregnancy because of the presence of non-
RBC stimulated ABO Abs.
ABO incompatibility ……
 Red blood cell destruction by ABO Abs is more
common than by anti-D.

 Fortunately, most cases are sub clinical and do not

necessitate treatment.

 Some infants may experience mildly elevated bilirubin

levels and some degree of jaundice with in the first
few days of life.

 These cases can usually be treated with phototherapy.

ABO incompatibility
 Mild red blood cell destruction, despite high
levels of maternal antibody occurs because:
 A or B substances are present in the fetal tissues and secretions
and bind or neutralize ABO antibodies, reduces the amount of ABO
Ab available to destroy fetal RBCs.

 Poor development of ABO Antigens on fetal or infant red blood cells.

 Presence of reduced number of A and B antigen sites on fetal or

infant red blood cells.

This also explains why the DAT is only weakly positive in most cases of
ABO HDN.
 Other blood group incompatibility
 Alloantibodies causing HDN other than anti-D
 Other Rh-system antibodies are known to cause HDN alone or in
combination with anti-D.

Kell HDFN (anti-K): Very severe at any titer

 Occurrence of disease is 1 in 1000 pregnancies

 88% of Kell HDFN patients have history of previous transfusion

 Major cause of fetal anemia is increased red cell destruction and
suppression of erythropoiesis

 Where K antigen is present on early erythroblasts

 anti-K causes immune destruction of early erythroid precursors so
women should be assessed immediately
 Others cont…..
Anti-c HDFN: 1:32 or greater identified in all affected fetuses
 0.7/1000 pregnancies occur

 40-50% mothers immunized by previous transfusion

 Only 20% of c positive infants require exchange transfusion

Anti-E HDFN: Most common after anti K, anti c and anti-D

 Antibody is often naturally occurring

Anti-k HDFN: this is very rare

 Despite the lower titer, be very severe

Others include:
 Anti-e, anti-Fya, anti-Jkb, and anti-M
Patho-physiology and clinical
presentations
 Increased destruction of red cells= mild to severe ANEMIA

 Enlargement of organs= Hepato-splenomegaly

 Osmotic and oncotic pressure disturbance= Hydrops fetalis

 Risk of labor: Asphayxia, splenic rupture

 Increase in unconjugated bilirubin= Mental retardation,

Kernicturus and brain damage

 Postnatal problems include: Asphyxia, pulmonary

hypertension, Pallor, Edema, Respiratory distress,
coagulopathies (decreased platelets and clotting factors)
 Hydropes fetalis

 Generalized EDEMA

 Hepato and
Splenomegaly
 Clinical features of HDFN

Clinical Features Rh ABO

Frequency Unusual Common
Anemia Marked Minimal
Jaundice Marked Minimal to moderate
Hydrops Common Rare
Hepatosplenomegaly Marked Minimal
Kernicterus Common Rare
 Common laboratory findings
 Mild to severe ANEMIA

 Hyperbilirubinemia= UNCONJUGATED and total

 Reticulocytosis (6-40%)

 Highly increased nucleated RBCs= ERYTHROBLASTOSIS

 Thrombocytopenia
 Leaukopenia

 DAT positive
 Hypoalbuminemia= EDEMA

 On smear:
Polychromasia, Anisocytosis, Increased NRBCs, Increased Spherocytes
Erythroblastosis fetalis
 High number of
Erythroblasts

 Anemia
Assessment of HDFN
Post partum testing
 It is advantageous to collect a sample of cord blood
from every new born.
 The specimen should be properly labeled and stored
for up to 7 days

 For testing if the mother is D Negative or if the new

born develops signs or symptoms of HDFN.

 Cord blood should be washed free of Wharton's jelly.

 Assessment…..
Post partum testing of infants Born to D-Negative Mothers

 Infants should be tested for the D antigen, including a test

for weak D

Postpartum Testing of infants with suspected HDFN

– Is based on medical history, physical examination of the new
born, and results of Laboratory testing on both mother and
child
– ABO, Rh and DAT were performed
– Most cases will have a positive DAT
• If DAT positive, perform elution to ID antibody
Assessment…..

ABO Testing
 In cases of HDFN the DAT may be positive,
which can lead to false positive or false
negative Rh- testing results.

 False Rh- negative results may be due to a

blocking phenomenon requiring gentle
elution to resolve.
Assessment…..
DAT
 Its result may be weak especially in cases of ABO
HDFN.
 When positive, performing an elution is optionalif Ab
identification test were performed on the mother at
the time of admission.
 If a maternal sample is unavailable, testing the elute
may be useful to confirm HDFN.
 If the maternal Antibody screen is negative and the
DAT is positive, ABO HDN should be suspected.
Assessment…..
 ABO HDN can be confirmed by performing either a heat
or freeze thaw elutes.

 The elute should be tested against A1, B and O cells

using an antiglobulin technique

 Positive results with A1 and/or B cells and negative

results with the O cells is indicative and /or ABO HDN.

 If the elute is negative with all cells, an antibody to a low

frequency antigen should be suspected and maternal
serum tested against the paternal cells.
 Comparison of laboratory features
Laboratory Features Rh ABO
Blood type of Mother Rh negative O
Blood type of Infant Rh positive A or B
Anemia Marked Minimal
Direct Coomb’s test Positive Negative/weak
pos??
Indirect Coomb’s test Positive Usually positive

Hyperbilirubinemia Marked Variable

RBC morphology Nucleated RBC Spherocytes

Treatment and Prevention : Rho (D)
Immune Globulin (Human)-RhIG
 Most effective immunological intervention is prevention of HDFN by
Rh immunoglobulin

 Rh Ig is a pooled IgG purified product

 Contains polyclonal IgG specific to the Rh blood group system

 IgG is directed against D polypeptide = Highly Immunogenic Antigen
 Administered

 To non-immunized Rh- negative mothers

 Who deliver Rh- positive babies

 Use
 Prevent Rh- alloimmunization.
Prevention……
 Candidates for this prophylaxis are:
 Mothers who are Rh-negative and Du - negative, and
 Have an Rh-positive or Du positive new born.

 All Rh-negative women who have abortions are candidates unless

the father or fetus is known to be Rh-negative.

The following are not RhIG candidates:

 Rho-negative women
 Who deliver Rh-negative babies.
 Whose serum contains anti-Rh (D).
 Who are Rh- positive or Du- positive
 Prevention RhIG …….
 Is a concentrate of IgG anti-D prepared from pools of human plasma.

 Is given intramuscularly

 To non-sensitized D-negative women at 28 weeks of gestation (ante partum) and

 Again within 72 hours of delivery (post partum) of a D positive infant.

Mechanism of action

 Suppresses the immune response following exposure to D positive fetal red

blood cells and prevents the mother from producing anti-D.

 Saturation of trophoblast Fc receptors, resulting in lower placental

transfer of maternal antibody.

 Reduced hemolysis of IgG coated red cells by fetal RES

Fetomaternal hemorrhage detection
 Accurate detection and quantification of fetal
RBCs in maternal circulation is necessary for
prevention

 Called calculating fetomaternal hemorrage

 FMH testing is also used in Obs/Gyn to determine

occult bleeding

 The commentially available Rh Ig have been

shown to be effective in prevention of HDFN
Prevention RhIG …….
 Before FMH screening and prophylaxis, death of
infants where 1.2/1000 births

 After the introduction of IgG Ig death dropped

to 0.02/1000 births

 One vial (300µg) of RhIg is sufficient to protect

against 30 ml of FMH
Estimation of FMH

 Rosette test

 Kleihäuer and Betke test

 Flow cytometry

Rosette Test:Qualitative test

Purpose
 To detect the presence of Rh positive RBCs in the
circulation of Rh negative person.

 to detect FMH > 30 ml

Rosette …

Principle
 Fetal Rh+ve RBCs in the maternal sample
react with the anti-D. The unbound antibody
is washed away and a suspension of group O,
Rh+ve cells is added.
 The anti-D reacts with both the Rh+ve and
the fetal Rh+ve RBCs. The RBCs agglutinate
in a rosette pattern, and the suspension is
examined microscopically.
Rosette Test - Procedure
 Use EDTA mother’s sample drawn after delivery

 Wash cells, add chemically modified anti-D and

enhancement

 Incubate 37oC

 Wash four times with normal saline

 Add R2R2 cells

 Centrifuge, mix, read microscopically

Rosette Test
 A qualitative measure of fetomaternal
hemorrhage

Fetomaternal Hemorrhage:
<1 rosette per 3 lpf = 1 dose of RhIg
>1 rosette per 3 lpf = Quantitate bleed
Kleihauer-Betke acid elution(Acid
Elution Stain)
 Quantitates the number of fetal cells in
circulation (To detect the presence of Hgb F)
 Fetal hemoglobin is resistant to acid and retain
their hemoglobin (appear bright pink)
 Adult hemoglobin is susceptible to acid and leaches
hemoglobin into buffer (“ghost” cells)
Acid Elution Stain- Purpose
Kleihäuer and Betke Test
 Red cells containing HbF resist acid elution
more than red cells containing HbA.
 On staining and counterstaining fetal cells
appear as darkly-stained red cells with a
background of palely-stained ghost red cells.
Acid Elution Stain Procedure
 EDTA sample

 Make a slide

 Fix smear

 Treat with acid

 Stain with Eosin

 Count number of stained HbF cells within 2000

HbA cells
Acid Elution Stain ……
Acid Elution Stain Calculations

 # fetal cells/2000 adult cells x 100 = %of fetal

cells

 % of fetal cells X 50 = number of mLs of fetal

bleed
Acid Elution Stain ……
Determine # vials RhIG
 FB/30X 2 = # vials needed

 300 μg RhIG will neutralize 30 mL of D+ whole blood

 Round the calculated dose up and add one more

vial for safety
 If # calculated is <0.5 round down, > or =0.5 round up
▪ 1.4 round down to 1 and add 1 vial=give 2 vials
▪ 1.6 round up to 2 and add 1 vial – give 3 vial
 Maternal Specimen

D Positive D negative

Anti-D present* Cord Blood

Mother not a
D Negative D Positive
candidate for RhIg

Rosette test:
Screens for FMH

Negative Positive
* Make sure presence of anti-D is
not due to antenatal administration
of Rh immune globulin One vial of RhIg Kleihauer-Betke:
within 72 hours quantitative

Calculate dose of
RhIg
Rh Immun0globulin dose
 Recommended dose (contained in one vial) is about
300 µg

 Offers protection against a feto-maternal

hemorrhage (FMH) of 30 ml (15 ml packed cells) or
less.

 If a massive FMH has occurred, the volume of the

hemorrhage must be determined to calculate the
number of vials to administer.
Flowcytometry
 Fluorescent markers attached to HbF
containing RBCs

 Mother’s blood treated with anti-D

 Fluorescein labelled anti-IgG added

 Detects down to D+ve cell in 1000 RBCs

 Treatment and laboratory testing

Treatment of mild anemia:

Phototherapy: treatment of choice for mild HDFN

jaundice
 Decomposes bilirubin into non toxic isomer
called photobilirubin, then liver excretes

 Blue light at wave of 420-470nm

 Soft eye shields are placed on baby to protect
eyes of baby
 Treatment…..
Treatment in severe cases:

Exchange transfusion:
 Involves continues removal of small amount of blood from
neonate with simultaneous infusion of donor blood until a
one or two-volume exchange is accomplished

Importance
 Removes sensitized cells and helps to reduce incomplete
maternal antibody
 Removes ~60% bilirubin, lowers bilirubin
 Provides infant with compatible red cells
Treatment.....
 is a continuous removal of small amounts of blood
from the neonate with simultaneous continuous
infusion of donor blood until a one or two-volume
exchange is accomplished.

 To give exchange transfusion to an infant clinical and

laboratory findings must be considered.

 Cord Hemoglobin (<10g/dl) and

 Raised serum bilirubin are strong indicators for

treatment.
Treatment.....
 For compatible exchange transfusion, donor’s blood
should be:

 Cross-matched with the maternal serum and

 Lack the RBC Ag corresponding to the maternal Abs
 ABO group and Rh type compatible with the infant’s blood
group.

 If the mother’s antibody is not available, group

O Rh negative red blood cells must be selected.
 Treatment…..
Intrauterine transfusion

Practiced by transfusing the fetus inside the uterus

based on anemia, maternal antibody and amniotic
fluid analysis
Treatment…..

Antenatal treatment of HDFN

 Plasma exchange for the mother

 Absorption of alloantibodies on to red cells
 Intravenous immunoglobulin given to mother
 Intrauterine transfusion
 Premature delivery or Induced labor
Treatment…..

Post natal treatment of HDFN

 Exchange transfusion to the fetus

 Phototheraphy to reduce serum

unconjugated bilirubin concentration

 Steroid therapy
Laboratory testing of HDFN
Prenatal laboratory investigation:

 Routine investigation is blood of the mother to

identify women at risk

 Once identified regular screening tests can be

used

 At time of booking ABO and Rh testing

 Antibody screening for the mother

 Testing…..
Antenatal investigations

Amniotic fluid analysis (Amniocentesis):

 Withdrawal of amniotic fluid

 Measure bilirubin and free Hgb concentration

 Both reflect the degree of destruction of fetal red

cells, severity of anemia and disease to the fetus
Testing…..
Chorionic villus sampling/biopsy;
 Cordocentesis
 Percutaneous umbilical cord blood sampling
(PUBS). Inserting needle into umbilical vein
under ultrasound guidance

Benefits:
 ABO and Rh(D) blood group, DAT, FBC (Hb),
 Film: Reticulocytes, Spherocytes,
Erythroblasts.
Testing…..
Ultrasound (Ultrasonography):
 Doppler technique is used as an aid in
intrauterine transfusion and amniocentesis

 Used diagnostically to detect symptoms of

hydrops, such as facial ascites, subcutaneous
edema or pericardial effusion

 High risk infants can be more effectively

monitored by Doppler ultrasound
Testing…..
Post natal investigation:
 Immediately after birth blood specimen from
the placental cord vein

Examine:
 ABO and Rh blood grouping/typing
 Direct anti-humanglobulin test (DAT)
 FBC/CBC, bilirubin, albumin and total protein
 Kleihauer and Betke test (acid elusion test)
ANY QUESTIONS!!!!!!!
Review Questions
1. Compare the causes and laboratory
demonstrating methods, of AIHA with HDN.
2. What is the cause of HDN? What consequences
could result from this condition?
3. What is exchange transfusion?
Review…..

4. Discuss the dose of IgG anti-D given to Rh-

pregnant women to prevent HDN?
5. When Rh HDN does occur?
6. List and discuss the postnatal laboratory
investigations to be carried out to know the
presence and extent of HDN.
Thank you !!!
Have a nice
day !!!