MLT 502 –

IMMUNOHAEMATOLOGY
II
HS221/5

GROUP C

HEMOLYTIC
DISEASE OF NEWBORN
(HDN)
 DEFINITION
A condition in which fetus or neonate’s red
blood cell (RBC) are destroyed by
Immunoglobulin G (IgG) antibodies
produced by mother.

 "hemolytic" means breaking down of

red blood cells
 "erythroblastosis" refers to making
of immature red blood cells
 "fetalis" refers to fetus
 HISTORY
Haemolytic disease of the newborn (HDN) has been known for
centuries with its typical and poor prognosis. The first
described by a French midwife who delivered twins in 1609.
One baby was swollen and died soon after birth, the other baby
developed jaundice and died several days later. For the next 300
years, many similar cases were described in which newborns
failed to survive.

During 1970s, the routine antenatal care included screening of

all expectant mothers to find those whose pregnancy may be at
risk of HDN, and giving preventative treatment accordingly.
This has led to decrease cases in the incidence of HDN,
particularly severe cases that were responsible for stillbirth and
neonatal death.
 CAUSES OF
HDN

Rh ABO OTHER SYSTEM

INCOMPATIBILIT INCOMPATIBILITY ANTIBODIES
Y INCOMPATIBILIT
Y

OTHER Rh
ANTIBODIES

OTHER ANTIBODIES
 CAUSES OF HDN
1) Rh incompatibility
-HDN is occured when a mother with Rh-negative blood
becomes pregnant with Rh-positive baby that inherited from
Rh-positive father.
-It occurs when anti-D is stimulated in mother plasma due to
mother ‘s immune response to the antigen D on fetal’s red
blood cells.
-This is due to anti-D is an IgG that capable to cross placenta
and hence delivered to fetal circulation.
- Rh caused HDN is less common but more severe.

2) ABO incompatibility
-HDN is arouse when a mother with blood type O becomes
pregnant with a fetus with different blood types A, B or AB.
- ABO antibodies is natural occurring that
antibodies
clinically significant.
3) Others unexpected immune antibody (other than anti-D) :
- Other Rh antibodies :
- anti-E (second most common, mild disease)
- anti–c (third most common, mild to severe)
- anti-C and anti-e (rare)
-antibody combination (anti-c and anti-E
occurring together, can be severe)
- Other antibodies
- Kell system antibodies (uncommon causes)
- Duffy, MNSs and Kidd system
antibodies (rare causes)

* Lewis and P system antibody are no occurrence in HDN.

 PHYSIOLOGIC OF HDN

Physiologic of HDN is defined as

process of how HDN occur
(mechanism).
 RHESUS HEMOLYTIC
DISEASE OF NEWBORN
• In a first pregnancy, Rh sensitization is not likely. Usually it
only becomes a problem in a future pregnancy with another
Rh positive baby.

• When the next pregnancy occur, the mother's antibodies

cross the placenta then reacts with an RBC antigen to fight
the Rh positive cells in the baby's body that the baby has
inherited from the father, and that is foreign to the mother.

• Hence, the antigen antibody interaction occurs.

• Sensitization of baby’s red blood cell (RBC) by mother’s IgG
antibody causes the baby’s RBC to be destroyed.

• These antibody-coated RBCs are removed from fetal circulation

by the macrophages of the spleen and liver.

• The severity of anemia depends on the amount of mother’s

antibody, its specificity, its avidity, and others characteristics.

• Anemia will stimulate bone marrow to produce more RBC

including immature RBC, which is then released to fetus
circulation. This is also known as erythroblastosis fetalis.
 This figure shows pathophysiology of HDN

Retrieved from: http://tmedweb.tulane.edu/pharmwiki/lib/exe/fetch.php/rhod.png

This figure explains the Mechanism of HDN
 ABO INCOMPATIBILITY OF
HDN
• ABO HDN is due to incompatibility of ABO between
mother and fetus.
• A portion of the mother’s ABO antibodies may be IgG
and cross the placenta.
• It thus sensitizes the fetal cells.
• This condition can occur on the first pregnancy and
can
occur without there having been transfusion.
• The mechanism of ABO HDN is similar to the RH
HDN.
• Occurrence of ABO HDN is somewhat common, but
fortunately it is not often very severe.
This figure shows circulating IgG maternal antibodies cross the
placenta and sensitize fetal antigens positive cells.

Retrieved from Essential of Immunohematology Book, John C. Flynn, Jr.

 • Once antigen-antibody interaction occurs, antibody-coated red blood
cells are removed from fetal circulation by macrophages of the spleen
and liver which lead to anemia
• Anemia limits the ability of the blood to carry oxygen to the baby's
ANEMIA organs and tissues.

• In response to anemia, fetal bone marrow and other hematopoietic

tissues in the spleen and liver increase the amount of red blood cells
production.
• Baby's responds to the hemolysis by trying to make more red blood
cells very quickly in the bone marrow and the liver and spleen.
ORGAN • Organs enlarge - hepatosplenomegaly.
ENLARGEME • New red blood cells released prematurely from bone marrow and are
NT
unable to do the work of mature red blood cells produced many
nucleated red blood cell which is unable to carry oxygen.

• As the red blood cells break down, bilirubin is formed

• Accumulation of bilirubin in body tissue and fluid results in jaundices.
• Due to immature liver of the newborn and inability to conjugated the
HYPERBILIR unconjugated bilirubin result in the unconjugated bilirubin increasing
UBINEMIA and crossing the blood brain barrier and cause kernicterus.
KERNICTERUS
• Kernicterus or bilirubin encephalopathy results from high
levels of unconjugated bilirubin in the fetus blood which
is more than 20 mg/dL .
• Because unconjugated bilirubin are lipid soluble and toxic ,
it
can crosses the blood-brain barrier and it will penetrates
neuronal and glial membrane thus cause neurotoxicity

• Patients surviving kernicterus have severe permanent

neurologic symptoms such as
a) choreoathetosis
b) spasticity
c) muscular rigidity
d) ataxia
e) deafness
f) mental retardation
Kernicterus

webhome.idirect.com/~brainology/braino http://www.nlm.nih.gov/medlineplus/ency/imagepages/1987
logy/devpath_25_kernicterus.html 5.htm

• Affected structures have a bright yellow color due to bilirubin stained .

 HYDROPS FETALIS
 As the fetus ‘s anemia worsen, the fetus
accelerates red cells production

Liver and spleen increase in size due to

increase production of red blood cells.

As a result, portal hypertension occur,

causing the liver to reduces its albumin
production.

 This in turn result in edema.

Edema is an abnormal accumulation of

fluid beneath the skin.

 This condition in the fetus is known as

hydrop fetalis

https://www.capefearvalley.com/outreach/Outreach/Peapods/
 CLINICAL SYMPTOM
• Varies from mild jaundice and anemia to hydrops fetalis
(with ascites, pleural and pericardial effusions)
• Chief risk to the fetus is anemia.
• Extramedullary hematopoiesis due to anemia results in
hepatosplenomegaly.
• Risks during labor and delivery include:
• asphyxia and splenic rupture.
• Postnatal problems include:
• Asphyxia
• Pulmonary hypertension
• Pallor (due to anemia)
• Edema (hydrops, due to low serum albumin)
• Respiratory distress
• Coagulopathies (↓ platelets & clotting factors)
• Jaundice
• Kernicterus (from hyperbilirubinemia)
• Hypoglycemia (due to hyperinsulinemnia from islet cell
hyperplasia)
 Test Situation Timing
Pregnancy Initial visit
ABO Typing

Pregnancy Initial visit & 26-28

Rh Typing
weeks
All Pregnancies Iniital visit
D negative pregancy Before RhIg therapy
Antibody Screen 3rd trimester if history
D positive pregnancy of antibodies or
transfusion
Antibody ID Positive antibody screen Upon detection
Upon initial detection

a
Rh or other clinically Repeat at 18 – 20
weeks
Prenat l Testing
Antibody titer
signficant antibody Repeat at 2-4
week intervals if
below critical titer
 POSTNATAL TESTING

• Determination of maternal ABO and D typing at

delivery.
 AMNIOCENTESIS
• Technique use to provide a specimen to assess the severity of
fetal hemolytic disease.

• A needle is introduced through abdomen and uterus and into the

amniotic sacs. A small amount of amniotic fluid is remove for
testing.

• Liley’s graph is use to evaluate the level of bile pigment in the

amniotic fluid to measure hemolysis in the fetus.

• Liley’s graph is used to correct for gestation of less than 27 week

because bilirubin level is normally peak at 23 to 25 week of
gestation in unaffected fetus.
 Liley’s Graph
Liley’s bottom zone represent affected or very mildy
affected fetus. The middle zone show minimally
affected fetus and the upper zone indicate moderate
to marked hemolysis in fetus. Higher concentration
of bilirubin in amniotic fluid is found in abnormal fetus.
 Management of
HDN

Treatment Prevention

Rh Immune
Globulin (RhIG or
Maternal Infant Rhogam)

Plasma Intra- Intrauterine Exchage Photo-

Exchange transfusion
venous transfusion therapy
Immune
Globulin
 TREATMENT
Intrauterine Transfusion (IUT)
• Red Blood Cells (RBCs) are infused into abdominal cavity of fetus and then absorbed
into fetal circulation to avoid hydrops fetalis and fetal death.
• Can be done as early as 17 weeks.
• In intraperitoneal IUT, a needle is passed through the mother’s abdomen and into the
abdomen of the fetus by the help of ultrasound image to determine the position of the
fetus and placenta.
• Selection of Blood for IUT:
- Most IUTs are accomplished using group O.
- Rh-negative RBCs that are less then 7 days from collection
- Ctyomegalovirus (CMV) antibody negative
- Hemoglobin S negative
- Fresh blood is used to provide RBCs with longest viability
and to avoid lower pH, decreased 2,3-diphospoglycerate, and
elevated potassium levels.
-RBCs are usually dry packed to remove residual anti-A and anti-B and reconstituted
with group AB fresh frozen plasma t provide coagulation factors.
 Intrauterine Transfusion (IUT)
• The risk of these procedures is now largely dependent on the prior condition of the
fetus and the gestational age at which transfusion is commenced.
• Titer greater than 32 for anti-D and 8 for anti-K OR four fold increase in titer
indicates need for analysis of amniotic fluid.
• Amniocentesis
• Perform at 28 wks if HDN in previous child
• Perform at 22 wks if previous child severely affected
• Perform if maternal antibody increases before 34th wk.
• High values of bilirubin in amniotic fluid analyses by the Liley method or a
hemoglobin concentration of cord blood below 10.0 g/mL.
Liley Chart
IUT Procedure
 Postpartum - Phototherapy
• Phototherapy is the treatment of choice.
• Phototherapy accelerates bilirubin metabolism through
the process of photodegradation .
• The effective wavelength of light seeems to be in te 420- to
475 nm range.
• During photoisomerization or photooxidation, the insoluble form
of unconjugated bilirubin is converted into water soluble form
which permits more rapid excretion without conjugation, through
the bile or urine.
 Phototherapy

• However phototherapy may not be effective in

controlling hyperbillirubinemia secondry to HDN if
the concentration of bilirubin rises at rate of 0.5 to
2.0 mg/dl per hour. In the cases, exchange
transfusion must occur.
 Exchange transfusion
Objective:
1. To decrease the level of bilirubin and prevent kernicterus.
2. To remove baby’s sensitized red blood cells.
3. To provide compatible red blood cells adequate oxygen carrying
capacity.
4. To decrease the level of incompatible antibody in the baby.
• Full-term infants rarely require an exchange transfusion if
the infant undergo the phototherapy.

• The exchange transfusion is done if the total bilirubin level

is aprroaching 20mg/dL and continues to rise despite the
baby undergo the phototherapy.

• The blood should be reconstituted from fresh, O negative

packed RBCs cross-matched against the mother and type-
specific fresh frozen plasma.

Continue….
 Technique uses:
• 30 min before the exchange transfusion, give albumin 1g/kg
to increase the bilirubin bound to albumin in the circulation
and make the exchange transfusion more effective.
• Exchange 2 times the blood volume, at 85mL/kg, by using
the isovolumic technique. This technique done by
withdrawing blood from upper atrium canal and infusing
through upper ventricle canal(low right atrium with tip intra
ventricle canal).
• Do not infuse blood through upper ventricle canal if tips is in
portal circulation.
• The blood should be warmed and the bag agitated every few
minutes to prevent settling of the RBCs.
 Plasma Exchange
• It is widely used treatment of immune-mediated disease.
• This treatment is applied to the pregnant women with
high antibody titer, or that has past history of stillbirth due
to HDN.
• This procedure are effective in decreasing the antibody
titer and quantity of antibody.
• Plasma exchange can reduce antibody titer up to 75%.
• Beside that, it also use for a way to delay the need for
fetal intervention that has been hydrops fetalis (edema) in
which before 22 week gestation in a previous pregnancy.
 Intravenous Immune Globulin
• Intravenous immune globulin (IVIG) is made up from
plasma isolated
• This treatment use strengthen body immune system
beside to treat immune deficiency
• Intravenous Immunoglobulins were found to
decrease hemolysis leading to reduction in serum
bilirubin level.
• The immunoglobulin could act by occupying the FC
receptors of reticulo-endothelial cells preventing
them from taking up and lysing antibody coated
RBCs. This subsequently leads to decrease in the
need for exchange transfusion.
 PREVENTION
Rh Immune Globulin (RhIG or Rhogam)
• Alloimmunization in pregnant woman can be prevented
by administration of Rh immune globulin (RhIG or
Rhogam).
• RhIG is a concentrate of IgG anti-D prepared from pooled
human plasma of D-negative people who have been exposed to
the D antigen and who have made antibodies to it.
• Significance of administration of RhIG
• (i) RhIG prevents alloimmunization in D-negative mother
exposed to D-positive fetal red cells. In the meantime, it protects
mother from being sensitized to D antigen of fetal during
pregnancy and after delivery of infant.
• (ii) RhIG suppresses mother’s immune response
following exposure to D-positive fetal red cells.
• (iii) RhIG prevents the mother from producing anti-D.
• (iv) RhIG protects subsequent D-positive pregnancies but
• Principle
 RhIG is given by injection into mother’s
muscle (intramuscular).
 Giving RhIG to D-negative pregnant woman prevents
her immune system from producing its own anti-D (which
would attack her D-positive fetal red cells).
 When RhIG is introduced into D-negative mothers’s
bloodstream, RhIG antibodies will locate D-positive fetal red
cells.
 RhIG antibodies will attach to D-positive fetal red cells
and covering their presence from mother’s immune
system.
 Therefore, sensitization of anti-D to D-positive fetal red
cells
can be prevented.
Retrieved from http://www.carolguze.com/text/102-13-humangenetics.shtml
• Guidelines for RhIG administration in D-negative pregnant woman :
(i)All doses should be given within 72 hours of delivery or procedure. If she is
not given RhIG within 72 hours after the birth of an Rh positive baby, she will
begin to make antibodies to the fetal blood cells.

(ii) Woman should not be sensitized to D antigen.

iii) 50 µg dose - Up to 12th week of gestation for abortion, miscarriage and end
period of ectopic pregnancy.

(iv) 120 µg dose

- End period of pregnancy which is after 12th week of gestation.
- After amniocentesis.
- After delivery of D-positive infant.

(v) 300 µg dose

- At 28th week of gestation
- After amniocentesis
- After delivery of D-positive infant.
 REFERENCES
• Quinley, E. D. (1998). Hemolytic Disease of Newborn. In
E. D. Quinley, Immunohematology Principle and
Practice (pp. 282-283). Philadelphia: Lippincott-Raven.
• Quinley, E. D. (1998). Hemolytic Disease of
Newborn. In E. D. Quinley, Immunohematology
Principle and Practice (pp. 282-283). Philadelphia:
Lippincott- Raven.