Download MBBS Transfusion Medicine and Blood Bank Presentations 2 Coombs Test Hdn Lecture Notes

Download MBBS (Bachelor of Medicine, Bachelor of Surgery) 1st Year, 2nd Year, 3rd Year and Final year Transfusion Medicine and Blood Bank 2 Coombs Test Hdn PPT-Powerpoint Presentations and lecture notes




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 cel s 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

? Al oimmunity

? Drug-induced immune-mediated mechanism.

Examples of al oimmune 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 cel s 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 cel s with IgG and/or complement

1. Compatibility testing.

2. Unexpected antibodies in serum.

Indirect antiglobulin test


Blood Sample

Blood Sample

fresh
Plain vial

Procedure:

2-3 drops of the test serum

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.

Centrifuge at 1000 rpm for 1 minutes.

Examine for hemolysis and/or agglutination-complete antibodies.

If not wash cells 3-4 times

Add 2 drops of AHG serum to the cells.

Mix and centrifuge at 1000 rpm for 1 minutes immediately.

Examine for agglutination- incomplete antibody
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

cel s having

blood cel s 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

cel s

antigens

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

Results in anemia

As the red blood cells break

down, bilirubin is formed

Baby's responds by trying to

make more red blood cells

Hyperbilirubinemia results

in the bone marrow, liver

in jaundice

and 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

? Pal or (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 cel s

? 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 cel s

? Reduces level of maternal antibody.

? Removes about 60 percent of bilirubin from the plasma

? Correct anemia

? Restores albumin and coagulation factors

This post was last modified on 08 April 2022