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HEM 2133

Immunohaematology I
Lesson 12:
Hemolytic Disease of Newborn II

Laboratory Investigation
Cord blood from infants born to Rh-negative
mothers should be tested for the D antigen
An Rh-negative woman with an Rh-positive
infant should receive one full dose of RhIG
within 72 hours of delivery, unless she is
known to be alloimmunized to D already
The presence of residual anti-D from
antepartum RhIG does not indicate ongoing

Neonatal studies
Cord blood samples should be collected from
every newborn and stored for at least 7 days
in the transfusion service in the event the
newborn shows signs of HDN
If HDN is suspected, both cord and maternal
blood should be tested for ABO and Rh (and
weak D if apparently D negative)
The maternal blood should also be tested for
unexpected antibodies if the mother is D
negative and the infant is D positive

A DAT should be performed on the cord blood

RBCs and an elution performed if the DAT is
positive and the antibody identified

Serologic Problems Seen in HDN

Cord blood should be washed several times
before testing
Often, cord blood is contaminated with
Whartons jelly which may cause false
(Whartons jelly - a soft connective tissue that
occurs in the umbilical cord)
If there is doubt as to the validity of
agglutination, a maternal sample should be
tested for antibody or a neonatal sample should
be tested for DAT and ABO/Rh

ABO/Rh Testing
Newborns who were transfused while
intrauterine often type Rh negative or weakly
Rh positive because so much of their
circulating RBCs are of donor origin
ABO grouping also reflects the donors ABO
group and may exhibit mixed-field reaction
with ABO antisera or, if the HDN is quite
severe , may group as an O

If the infants RBCs are heavily coated with IgG

antibodies, the Rh typing may give either
false-positive or false-negative results
The use of saline antisera or chemically
modified antisera may help in obtaining a true
Rh type

Direct Antiglobulin Test

The strength of the DAT varies depending on:
The strength of the maternal antibody
The number of antigenic sites for the antigen
to which the maternal antibody is directed
How much blood the infant has received from
If the maternal blood is available and a single
antibody is identified, an elution from the
infants RBCs is unnecessary

If however, the maternal serum is negative for

unexpected antibodies, ABO antibodies or an
antibody to a low-frequency antigen must be
Antiglobulin testing of the eluate from the infants
cells against A1, B and O cells aids in the diagnosis
Testing the eluate against paternal RBCs aids in
diagnosing a low-frequency antibody
In addition, testing the maternal serum against the
paternal RBCs confirms the presence of an
antibody even when antibody screening tests and
panels are negative

Postnatal Testing Required If HDN is

Mothers blood
Rh including weak D if D-negative
Indirect antiglobulin test (antibody screen)
If indirect antiglobulin test is positive, do
antibody identification test
If antibody identification test is positive for
IgG antibody, do hemoglobin and bilirubin on

Cord blood
ABO forward only
Rh including weak D if D-negative
Direct antiglobulin test
If direct antiglobulin test is positive, do elution
and antibody identification test on eluate

Treatments for HDN

In utero
Intrauterine transfusion (IUT) can occur either
by the intraperitoneal route or the direct
intravascular approach by the umbilical vein
In intraperitoneal IUT, a needle is passed
through the mothers abdomen and into the
abdomen of the fetus
RBCs are infused into the abdominal cavity of
the fetus and then absorbed into the fetal

Intrauterine Transfusion

Selection of blood for intrauterine transfusion

Most IUT are accomplished using
Group O, Rh-negative RBCs that are less than
7 days from collection
Cytomegalovirus antibody negative or
Gamma irradiated
Hemoglobin S negative

Group O, Rh-negative RBCs are used because the

ABO group and Rh type of the fetus are usually
Fresh blood is usually used to provide RBCs with
the longest viability and to avoid lower pH,
decreased 2,3-diphosphoglycerate and elevated
potassium levels
Primary CMV infection in a fetus or newborn has
the potential for causing death
CMV antibody-negative or leukoreduced blood is
transfused to avoid this route of infection

Gamma irradiation of cellular products is

necessary to prevent graft-versus-host disease in
the fetus
Hemoglobin S-negative blood is used because the
decreased oxygen tension that may occur during
gestation may cause hemoglobin S-containing
blood to sickle
RBCs are usually dry packed to remove residual
anti-A and anti-B and reconstituted with group
AB fresh frozen plasma to provide coagulation

Two consequences of HDN that may require
intervention and treatment after birth are
hyperbilirubinemia and anemia
High level of free unconjugated bilirubin are
associated with neurotoxicity
Treatment modalities for hyperbilirubinemia
are phototherapy sessions or exchange

Accelerates bilirubin metabolism through the
process of photodegradation
Effective wavelength = 420-475 nm
The insoluble form of unconjugated bilirubin is
converted into a water-soluble form, which
permits more rapid excretion, without
conjugation, through the bile or urine


Exchange transfusion
When the serum bilirubin reaches a level of 18 to
20 mg/dL in any infant with HDN, exchange
transfusion must be performed
The coated infant RBCs are removed and replaced
with RBCs with normal survival
This reduces the potential for increased bilirubin
as well as reducing some of the bilirubin already

The number of unbound antibody molecules

available to attach to newly formed antigenpositive cells is also reduced
Exchange transfusion is performed in 45 to 90
minutes by using 5 to 10 ml increments of
The increment of the infants blood is
removed first and replaced by the donor
blood in an equal volume

Selection of blood
The RBCs used in an exchange transfusion are
from a donor whose blood type is compatible
with the mother and infants serum
It is estimated that an exchange transfusion
with a volume double that of the infants
blood volume replaces approximately 85% of
the infants circulating RBCs

Because bilirubin production still may remain

high, the immediate decrease in bilirubin
concentration is usually only 50%
It is often followed by a rebound rise in
bilirubin that may necessitate additional
exchange transfusion, usually in conjunction
with phototherapy
Furthermore, bilirubin and the offending
antibody are distributed throughout the
extracellular space and thus reenter the
vascular space after exchange transfusion

In addition, tissue-bound bilirubin

reequilibrates with extravascular components,
reentering the circulation and contributing to
the rebound
Premature newborn infants are more likely
than full-term infants to require exchange
transfusion for elevated bilirubin because
their livers are less able to conjugate bilirubin

Transfusions and compatibility testing

Infants with HDN , as well as many premature
infants, require ongoing transfusions to replace
blood lost through normal physiologic processes
and blood sampling for laboratory tests
The initial pretransfusion sample must be tested
for ABO and Rh (and for weak D if the infant is D
The ABO needs only forward grouping because
ABO antibodies are not developed yet

If other than group O RBCs are to be

transfused, an indirect antiglobulin test with A
and B RBCs must be performed
If residual anti-A or anti-B is present, blood of
those groups must be avoided until they are
no longer demonstrable
An antibody screen must be performed
The serum or plasma of the mother or infant
may be used for antibody screen

Once these tests have been performed, repeat

testing for ABO and Rh may be omitted for the
remainder of the infants hospital stay
If the antibody screen is negative,
crossmatching is not required for the initial or
subsequent transfusions
These procedures apply to all infants younger
than 4 months of age

If an unexpected antibody is present, RBCs for

transfusion must lack the corresponding
antigens or be crossmatch compatible by the
antiglobulin technique until the antibody is no
longer present in the infants serum