Objectives
?
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Understand types of Coomb`s test
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Indications, Steps and interpretation?
Hemolytic Disease of the Newborn
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State the testing to be performed on the mother to monitor the
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severity of HDN.?
List the laboratory tests and values
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State the treatment options
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?State the requirements of blood to be used for transfusion of the
fetus and newborn.
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Antigen antibody reactions
ANTIGLOBULIN TEST
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? Detection of antibodies- (IgG or
complement) affixed to RBCs or free in
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plasma? in vivo-Direct antiglobulin test (DAT)
? in vitro -Indirect antiglobulin test (IAT)
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Types of Coomb`s Test
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DAT- Direct antiglobulin test
IAT- Indirect antiglobulin test
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ANTIGLOBULIN TEST? Principle - Antihuman globulins (AHG) bind
to human globulins either free in serum or
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attached to RBCs
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ANTIGLOBULIN TEST
? Pentameric IgM Abs are so
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large that, when bound toRBC Ags, the RBCs
agglutinate (usually at RT)
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? IgG Abs usually need a little
help, a bridge molecule, to
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agglutinate RBCs? AHG acts as a bridge
molecule
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The Antiglobulin Test
Antiglobulin serum (Coombs'Serum) was
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discovered by Coombs etal in 1945.
Anti-Human Globulin (AHG) Reagent
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? Preparation? Anti-human globulin reagent is
produced by immunizing rabbits,
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goats or sheep with human serum
or purified type antigen.
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? Animals are bled after a specifiedperiod and the reagent is purified
by absorbing unwanted antibodies.
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Types of AHG reagent
Polyspecific antiglobulin reagent
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human IgG, C3 and C4
Monospecific antiglobulin reagent
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Any one- human IgM, IgD, IgA,C3 or C4
DIRECT ANTIGLOBULIN TEST (DAT)
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DAT? detects sensitized red cel s with IgG and/or
complement components C3b and C3d in vivo.
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? In vivo coating may occur when any immune
mechanism is attacking the patient's own RBC's.
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? Autoimmunity? Al oimmunity
? Drug-induced immune-mediated mechanism.
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Examples of al oimmune hemolysis
? Hemolytic transfusion reaction
? Hemolytic disease of the newborn (also known as
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HDN or erythroblastosis fetalis)
? Rhesus D
? ABO
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? Anti-Kell? Rhesus c, E
? Other -RhC, Rhe, Kidd, Duffy, MN, P or others
Examples of autoimmune hemolysis
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? Warm antibody autoimmune hemolytic anemia? Idiopathic
? Systemic lupus erythematosus
? Cold antibody autoimmune hemolytic anemia
? Infectious mononucleosis
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? Paroxysmal cold hemoglobinuria (rare)Drug-induced immune-mediated hemolysis
? Methyldopa
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? Penicillin
? Quinidine
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? CephalosporinsBlood Sample
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Blood Samplefresh
EDTA vial
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Procedure of DAT1 drop of EDTA sample
Wash the red cel s 3-4 times in saline- to remove free globulin
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molecules.Add 2 drops of polyspecific AHG serum
Mix, Centrifuge at 1000 rpm for 1 minute
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Check for agglutination
Add Check (IgG coated) cells to a negative test. If agglutination is
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Indirect Antihuman globulin Test (IAT)Indications- to determine the presence of free
antibodies in serum.
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in vitro sensitization of red cel s with IgG and/or complement
1. Compatibility testing.
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2. Unexpected antibodies in serum.Indirect antiglobulin test
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Blood SampleBlood Sample
fresh
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Plain vialProcedure:
2-3 drops of the test serum
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Add 1 drop of 3-5% suspension of washed O Rh (D) positive red cells
Mix and incubate at 37?C for 30-40 minutes.
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Centrifuge at 1000 rpm for 1 minutes.Examine for hemolysis and/or agglutination-complete antibodies.
If not wash cells 3-4 times
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Add 2 drops of AHG serum to the cells.
Mix and centrifuge at 1000 rpm for 1 minutes immediately.
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Examine for agglutination- incomplete antibodyAntigen-Antibody Ratio
? Prozone - antibody excess: Antibodies saturating all
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antigen sites; no antibodies forming cross-linkagesbetween cells; no agglutination
? Zone of equivalence: antibodies and antigens
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present in optimum ratio, agglutination formed
? Zone of antigen excess (Post-zone): too many
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antigens - any agglutination is hidden by masses ofunagglutinated antigens
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COOMB'S CELLS? Antibody-coated cells are used as a positive
indicator
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? To show that test cells were properly washed
? No reagent deterioration has occurred
? Failure to agglutinate-test result is not valid
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Hemolytic Disease of the NewbornCause of Hemolytic Disease
Pregnancy with
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fetal red blood
Exposure to red
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cel s havingblood cel s during
antigen(of
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transfusion.
paternal origin)
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MaternalIgG
antibodies
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produced
Cause of Hemolytic Disease
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Antigen of
Maternal IgG
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paternal originantibodies cross
present on the
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the placenta to
fetal red blood
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coat fetalcel s
antigens
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Decreased red
blood cell
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survival whichcan result in
anemia
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Three Classifications of HDN? Rh ? anti-D
? ABO
? "Other" ?anti-C, c, E, e, Jk, K, Fy, S etc.
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Rh Hemolytic Disease
? Anti-D is the commonest form of
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severe HDN? mild to severe.
ABO Hemolytic Disease
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? Mother group O-anti-A, -B and ?A,B in theirplasma
? Fetal group A or B- RBCs attacked by
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antibodies
? Occurs in only 3%, is severe in only 1%
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"Other" Hemolytic Disease? Uncommon, occurs in ~0.8% of pregnant women.
? Anti-K
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? mild to severe
? usually caused by multiple blood transfusions
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? is the second most common form of severe HDNHemolysis of fetal red blood
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cellsResults in anemia
As the red blood cells break
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down, bilirubin is formed
Baby's responds by trying to
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make more red blood cellsHyperbilirubinemia results
in the bone marrow, liver
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in jaundice
and spleen
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New red blood cells releasedprematurely from bone
Hepatosplenomegaly
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marrow and are unable to
do the work of mature red
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blood cellsComplications During Pregnancy
? Severe anemia
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? Hydrops Fetalis
? Baby's organs are unable to handle the anemia
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? 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
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stillborn.
Postnatal problems
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? Asphyxia
? Pulmonary hypertension
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? Pal or (due to anemia)? Edema (hydrops, due to low serum albumin)
? Respiratory distress
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? Coagulopathies ( platelets & clotting factors)
? Jaundice
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? Kernicterus (from hyperbilirubinemia)? Hypoglycemia (due to hyperinsulinemnia from
islet cell hyperplasia)
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Kernicterus (bilirubin encephalopathy)
? High levels of indirect bilirubin (>20
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mg/dL)? crosses the blood-brain barrier-
unbound unconjugated bilirubin
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? penetrates neuronal and glial
membranes- lipid soluble
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? toxic to nerve cel s? Patients who survive kernicterus have
severe permanent neurologic
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symptoms
? Choreoathetosis, spasticity, muscular
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rigidity, ataxia, deafness, mentalretardation).
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Laboratory Findings? Anemia
? Hyperbilirubinemia
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? Reticulocytosis (6 to 40%)
? nucleated RBC count (>10/100 WBCs)
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? Thrombocytopenia? Leukopenia
? Positive Direct Antiglobulin Test
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? Hypoalbuminemia
? Rh negative blood type or ABO incompatibility
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? Smear: polychromasia, anisocytosis, no spherocytesMCA Doppler study
? Reliable non-invasive screening tool to detect fetal anemia.
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? The vessel can be easily visualized with color flow Doppler
as early as 18 weeks' gestation.
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? In cases of fetal anemia, an increase in the fetal cardiacoutput and a decrease in blood viscosity contribute to an
increased blood flow velocity
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Blood Bank TestingManagement
? Measure bilirubin in cord blood and at least every 4 hours
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for the first 12 to 24 hours
? Transcutaneous Monitoring
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Intrauterine Transfusion (IUT)
? To prevent hydrops fetalis and fetal death.
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? Transfusions done every 1 to 4 weeks until the fetus is mature enough tobe delivered safely.
? A compatible blood type (usually type O, Rh-negative) is delivered into the
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fetus's abdominal cavity or into an umbilical cord blood vessel.
Selection of Blood
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? 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.
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? The volume transfused- 75-175 mL depending on the fetal size
and age.
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? CMV negative? IRRADIATED
? O negative, lack all antigens to which mom has antibodies and
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Coomb's compatible.
Treatment of Mild HDN
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? Phototherapy is the treatment of choice.
Exchange Transfusion
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? If the total serum bilirubin level is approaching 20 mg/dL? Continues to rise despite intense in-hospital phototherapy.
? Removes
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? sensitized cel s
? Reduces level of maternal antibody.
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? Removes about 60 percent of bilirubin from the plasma? Correct anemia
? Restores albumin and coagulation factors
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