Coombs Test HDN

The Antiglobulin Test
Hemolytic disease of newborn

Objectives
• Understand types of Coomb`s test
– Indications, Steps and interpretation
• Hemolytic Disease of the Newborn
– State the testing to be performed on the mother to monitor the
severity of HDN.
– List the laboratory tests and values
– State the treatment options
– State the requirements of blood to be used for transfusion of the
fetus and newborn.
Antigen antibody reactions
ANTIGLOBULIN TEST
• Detection of antibodies- (IgG or
complement) affixed to RBCs or free in
plasma
– in vivo-Direct antiglobulin test (DAT)
– in vitro -Indirect antiglobulin test (IAT)
Types of Coomb`s Test
DAT- Direct antiglobulin test IAT- Indirect antiglobulin test
ANTIGLOBULIN TEST
• Principle - Antihuman globulins (AHG) bind
to human globulins either free in serum or
attached to RBCs
ANTIGLOBULIN TEST
– Pentameric IgM Abs are so
large that, when bound to
RBC Ags, the RBCs
agglutinate (usually at RT)
– IgG Abs usually need a little

help, a bridge molecule, to
agglutinate RBCs
– AHG acts as a bridge

molecule
The Antiglobulin Test
Antiglobulin serum (Coombs’Serum) was
discovered by Coombs etal in 1945.
Anti-Human Globulin (AHG) Reagent
• Preparation
– Anti-human globulin reagent is
produced by immunizing rabbits,
goats or sheep with human serum
or purified type antigen.
– Animals are bled after a specified
period and the reagent is purified
by absorbing unwanted antibodies.
Types of AHG reagent
Polyspecific antiglobulin reagent
human IgG, C3 and C4
Monospecific antiglobulin reagent
Any one- human IgM, IgD, IgA,
C3 or C4
DIRECT ANTIGLOBULIN TEST (DAT)
DAT
• detects sensitized red cells with IgG and/or
complement components C3b and C3d in vivo.
• In vivo coating may occur when any immune

mechanism is attacking the patient's own RBC's.
– Autoimmunity
– Alloimmunity
– Drug-induced immune-mediated mechanism.
Examples of alloimmune hemolysis
• Hemolytic transfusion reaction
• Hemolytic disease of the newborn (also known as
HDN or erythroblastosis fetalis)
– Rhesus D
– ABO
– Anti-Kell
– Rhesus c, E
– Other -RhC, Rhe, Kidd, Duffy, MN, P or others
Examples of autoimmune hemolysis
– Warm antibody autoimmune hemolytic anemia

– Idiopathic
– Systemic lupus erythematosus
– Cold antibody autoimmune hemolytic anemia
– Infectious mononucleosis
– Paroxysmal cold hemoglobinuria (rare)
Drug-induced immune-mediated hemolysis
• Methyldopa
• Penicillin
• Quinidine
• Cephalosporins
Blood Sample
Blood Sample
fresh
EDTA vial
Procedure of DAT
1 drop of EDTA sample
Wash the red cells 3-4 times in saline- to remove free globulin
molecules.
Add 2 drops of polyspecific AHG serum
Mix, Centrifuge at 1000 rpm for 1 minute
Check for agglutination
Add Check (IgG coated) cells to a negative test. If agglutination is

obtained, the result is valid.
Indirect Antihuman globulin Test (IAT)
Indications- to determine the presence of free
antibodies in serum.
in vitro sensitization of red cells with IgG and/or complement
1. Compatibility testing.
2. Unexpected antibodies in serum.
Indirect antiglobulin test
Blood Sample
Blood Sample
fresh
Plain vial
Procedure:
Antigen-Antibody Ratio
• Prozone - antibody excess: Antibodies saturating all
antigen sites; no antibodies forming cross-linkages
between cells; no agglutination
• Zone of equivalence: antibodies and antigens
present in optimum ratio, agglutination formed
• Zone of antigen excess (Post-zone): too many
antigens - any agglutination is hidden by masses of
unagglutinated antigens
COOMB’S CELLS
• Antibody-coated cells are used as a positive
indicator
– To show that test cells were properly washed
– No reagent deterioration has occurred
• Failure to agglutinate-test result is not valid
Hemolytic Disease of the
Newborn
Cause of Hemolytic Disease
Pregnancy with
fetal red blood Exposure to red
cells having blood cells during
antigen(of transfusion.
paternal origin)
Maternal IgG
antibodies
produced
Cause of Hemolytic Disease
Antigen of
Maternal IgG
paternal origin
antibodies cross
present on the
the placenta to
fetal red blood
coat fetal antigens
cells
Decreased red blood

cell survival which
can result in anemia
Three Classifications of HDN
• Rh – anti-D
• ABO
• “Other” –anti-C, c, E, e, Jk, K, Fy, S etc.
Rh Hemolytic Disease
• Anti-D is the commonest form of
severe HDN
• mild to severe.
ABO Hemolytic Disease
• Mother group O-anti-A, -B and –A,B in their
plasma
• Fetal group A or B- RBCs attacked by
antibodies
• Occurs in only 3%, is severe in only 1%
“Other” Hemolytic Disease
• Uncommon, occurs in ~0.8% of pregnant women.
• Anti-K
– mild to severe
– usually caused by multiple blood transfusions
– is the second most common form of severe HDN
Hemolysis of fetal red blood
cells
As the red blood cells break

Results in anemia
down, bilirubin is formed
Baby's responds by trying to

make more red blood cells in Hyperbilirubinemia results in
the bone marrow, liver and jaundice
spleen
New red blood cells released

prematurely from bone
Hepatosplenomegaly marrow and are unable to do
the work of mature red blood
cells
Complications During Pregnancy
• Severe anemia
• Hydrops Fetalis
– Baby's organs are unable to handle the anemia
– The heart begins to fail
– Fluid build up in the baby's tissues and organs
• A fetus with hydrops is at great risk of being

stillborn.
Postnatal problems
– 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)
Kernicterus (bilirubin encephalopathy)
• High levels of indirect bilirubin (>20
mg/dL)
– crosses the blood-brain barrier-
unbound unconjugated bilirubin
– penetrates neuronal and glial
membranes- lipid soluble
– toxic to nerve cells
• Patients who survive kernicterus have
severe permanent neurologic
symptoms
– Choreoathetosis, spasticity, muscular
rigidity, ataxia, deafness, mental
retardation).
Laboratory Findings
• Anemia
• Hyperbilirubinemia
• Reticulocytosis (6 to 40%)
• ↑ nucleated RBC count (>10/100 WBCs)
• Thrombocytopenia
• Leukopenia
• Positive Direct Antiglobulin Test
• Hypoalbuminemia
• Rh negative blood type or ABO incompatibility
• Smear: polychromasia, anisocytosis, no spherocytes
MCA Doppler study
• Reliable non-invasive screening tool to detect fetal anemia.
– The vessel can be easily visualized with color flow Doppler
as early as 18 weeks’ gestation.
– In cases of fetal anemia, an increase in the fetal cardiac
output and a decrease in blood viscosity contribute to an
increased blood flow velocity
Blood Bank Testing
Management
• Measure bilirubin in cord blood and at least every 4 hours
for the first 12 to 24 hours
• Transcutaneous Monitoring
Intrauterine Transfusion (IUT)
• To prevent hydrops fetalis and fetal death.
• Transfusions done every 1 to 4 weeks until the fetus is mature enough to
be delivered safely.
• A compatible blood type (usually type O, Rh-negative) is delivered into the
fetus's abdominal cavity or into an umbilical cord blood vessel.
Selection of Blood
• CPD, as fresh as possible, preferably <5 days old.
• A hematocrit of 80% or greater is desirable to minimize the
chance of volume overload in the fetus.
• The volume transfused- 75-175 mL depending on the fetal size
and age.
• CMV negative
• IRRADIATED
• O negative, lack all antigens to which mom has antibodies and
Coomb’s compatible.
Treatment of Mild HDN
• Phototherapy is the treatment of choice.
Exchange Transfusion
• If the total serum bilirubin level is approaching 20 mg/dL

• Continues to rise despite intense in-hospital phototherapy.
• Removes
– sensitized cells
– Reduces level of maternal antibody.
– Removes about 60 percent of bilirubin from the plasma
• Correct anemia
• Restores albumin and coagulation factors

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The Antiglobulin Test

Hemolytic disease of newborn

– IgG Abs usually need a little

– AHG acts as a bridge

• In vivo coating may occur when any immune

– Warm antibody autoimmune hemolytic anemia

Add 2 drops of polyspecific AHG serum

Mix, Centrifuge at 1000 rpm for 1 minute

Check for agglutination

Add Check (IgG coated) cells to a negative test. If agglutination is

Decreased red blood

As the red blood cells break

Baby's responds by trying to

New red blood cells released

• A fetus with hydrops is at great risk of being

• If the total serum bilirubin level is approaching 20 mg/dL

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