HEM 2133

Immunohaematology IImmunohaematology 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 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 protection

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 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 agglutination

(Whartons jelly - a soft connective tissue that (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 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 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 How much blood the infant has received from transfusions

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 suspected

Antiglobulin testing of the eluate from the infants cells against A1, B and O cells aids in the diagnosis of ABO HDN

Testing the eluate against paternal RBCs aids in 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

Suspected

Mothers blood

ABO

Rh including weak D if D-negative

Indirect antiglobulin test (antibody screen) 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 baby

Cord blood

ABO forward only

Rh including weak D if D-negative

Direct antiglobulin test

If direct antiglobulin test is positive, do elution 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 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 circulation

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

leukoreducedleukoreduced

Gamma irradiated

Hemoglobin S negative

Group O, Rh-negative RBCs are used because the

ABO group and Rh type of the fetus are usually

unknown

Fresh blood is usually used to provide RBCs with

the longest viability and to avoid lower pH,

decreased 2,3-diphosphoglycerate and elevated

potassium levelspotassium 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 RBCs are usually dry packed to remove residual anti-A and anti-B and reconstituted with group AB fresh frozen plasma to provide coagulation factors

Postpartum

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 neurotoxicityassociated with neurotoxicity

Treatment modalities for hyperbilirubinemia

are phototherapy sessions or exchange

transfusions

Phototherapy

Accelerates bilirubin metabolism through the

process of photodegradation

Effective wavelength = 420-475 nm 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

Phototherapy

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 This reduces the potential for increased bilirubin

as well as reducing some of the bilirubin already

formed

The number of unbound antibody molecules

available to attach to newly formed antigen-

positive cells is also reduced

Exchange transfusion is performed in 45 to 90

minutes by using 5 to 10 ml increments of minutes by using 5 to 10 ml increments of

blood

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 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 phototherapywith 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 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 The initial pretransfusion sample must be tested

for ABO and Rh (and for weak D if the infant is D

negative)

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 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, 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 antiglobulin technique until the antibody is no

longer present in the infants serum