Lab 5 Coombs Tests

8/11/2019 Lab 5 Coombs Tests

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Ant i -Globul in TestDirect , Indirect

PracticalBlood Bank


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The Antiglobulin Test Antiglobulin serum (Coombs Serum) was

discovered by Coombs in 1945.

The antiglobulin test can be used to detect red cellssensitized with IgG alloantibodies, IgG

autoantibodiesor complement components.

Sensitization of red cells can occur in vivo or vitro.

The use of AHG serum to detect sensitization of redcells in vitro can be:

One stage technique , the direct antiglobulin test(DAT).

Two stage technique , the indirect antiglobulintest (IAT).


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PRINCIPLE

Normal human red blood cells, in presence ofantibody directed towards the antigen they possess,may fail to agglutinate when centrifuged andbecome sensitized. This may be due to theparticular nature of the antigen and antibodyinvolved.

Sensitization of RBCs may be with IgG orcomplement.

In order for agglutination to occur an additional ofanti-antibody or anti-complements, which reactswith the Fc portion of the IgG antibody, or with theC3b or C3d component of complementalternatively.


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PRINCIPLE

This will form a "bridge" between the antibodies or

complement coating the red cells, causingagglutination.

The coating (sensitization) of red cells can occur invivo or in vitro following incubation at 37C with

serum containing antibody.


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Production Methods of Anti-Human globulin

(AHG or Coombs) Reagent

May be made by injecting rabbits , goats or sheepwith purified human IgG or C3, then harvesting theantibodies produced by the rabbit.

Monoclonal technology may be used to makemonoclonal antiglobulin reagent.


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Types of AHG reagent

Polyspecific Anti-human Globulin: blend of Anti-IgGand Anti-C3b, -C3d

Monospecific reagents: Anti-IgG alone or Anti-C3b,-C3d alone

Note: Reagent does not contain antibodies toIgM. Information about IgM coating of cells comesfrom the presence of C3 coating the cells since IgM is a

strong complement activator.


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DIRECT ANTIGLOBULIN TEST (DAT)


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DAT

The direct antiglobulin test (DAT) detects sensitized

red cells with IgG and/or complement componentsC3b and C3d in vivo.

In vivo coating of red cells with IgG and/orcomplement may occur in any immune mechanism

is attacking the patient's own RBC's. These mechanism could be:

Autoimmunity

Alloimmunity

Or a drug-induced immune-mediated mechanism.


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Examples of alloimmune hemolysis

Hemolytic transfusion reaction

Hemolytic disease of the newborn (also known asHDN or erythroblastosis fetalis)

Rhesus D hemolytic disease of the newborn (alsoknown as Rh disease)

ABO hemolytic disease of the newborn (theindirect Coombs test may only be weaklypositive)

Anti-Kellhemolytic disease of the newborn

Rhesus c, E hemolytic disease of the newborn

Other blood group incompatibility (RhC, Rhe,

Kidd, Duffy, MN, P and others)


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Warm antibody autoimmune hemolytic anemia

Idiopathic

Systemic lupus erythematosus

Evans' syndrome (antiplatelet antibodies and

hemolytic antibodies)

Cold antibody autoimmune hemolytic anemia

Idiopathic cold hemagglutinin syndrome

Infectious mononucleosis

Paroxysmal cold hemoglobinuria (rare)

Examples of autoimmune hemolysis


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Drug-inducedimmune-mediated hemolysis

Methyldopa (IgG mediated type II hypersensitivity)

Penicillin (high dose)

Quinidine (IgM mediated activation of classical

complement pathway and Membrane attackcomplex)


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Blood Sample

Whole Blood Sample - It should be as fresh aspossible not more than 24 hours old,

otherwise, the sample should be taken in EDTA.


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Procedure of DAT

1. Take 2-3 drops of blood to be tested in a clean labeled tube.2. Wash the red cells 3-4 times in a large volume of saline to remove

free globulin molecules. Remove all supernatant after each wash.

Completely decant the final supernatant wash.

3. Add 2 drops of polyspecific AHG serum in 1 drop of sensitized

washed red cells or in 1 drop of 3-5 % suspension of sensitized cellsimmediately.

4. Mix, Centrifuge at 1000 rpm for 1 minutes immediately.

5. Gently shake the tube to dislodge the cell button and see for

agglutination, use optical aid if needed, Record the result.6. Add 1 drop of IgG coated red cells to a negative test. Mix,

centrifuge at 1000 rpm for 1 min. Immediately look for

agglutination. If a negative result (no agglutination) is obtained the

test result is invalid and whole test should be repeated. If

agglutination is obtained, the result is valid.


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Indirect Antihuman globulin Test (IAT)

Indications The IAT is done to determine the presence of

sensitization of red cells with IgG and/orcomplement in vitro in the following conditions.

1. Compatibility testing.2. Screening and detection of unexpected

antibodies in serum.

3. Determination of red cells phenotype K, Lea, Fya

Fyb, Jka, Jkb and sub-group of Rh etc by usingknown sera.


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Indirect antiglobulin test


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Procedure:

1. Place 2-3 drops of the test serum in a tube. Serumshould be fresh for detecting complementcomponents and complement binding antibodies,otherwise, fresh AB serum should be added to it.

2. Add 1 drop of 3-5% suspension of washed O Rh (D)

positive red cells to the serum in the tube.3. Mix and incubate at 37C for 30-40 minutes.4. Centrifuge at 1000 rpm for 1 minutes.5. Examine for hemolysis and/or agglutination. Use

optical aid if necessary. Agglutination at this stage

indicates the presence of saline (complete)antibodies.

6. If no agglutination is seen, wash cells 3-4 times inlarge volume of saline. Decant supernatant in eachwash as completely as possible.

P d


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Procedure:

7. Add 2 drops of AHG serum to the cells.8. Mix and centrifuge at 1000 rpm for 1 minutes

immediately.9. Gently shake the tube to dislodge the button and

examine for agglutination, using optical aid.Record the result.

10. Add 1 drop of IgG coated red cells to any test thatis negative. Mix and centrifuge at 1000 rpm for 1minutes. Look for agglutination. If there is noagglutination, the test result is invalid and thewhole test is repeated. If agglutination is obtainedthe result is valid.

11. Auto control should be kept with IAT.


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BOVINE ALBUMIN(22%)-IAT

One Stage Method - Additive method

Procedure:1. Two drops of albumin 22.5% are added in step (2) of

saline-IAT

2. Mix and incubate for 20-30 minutes at 37C

3. Proceed further as in saline-IAT procedure.


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Antigen-Antibody Ratio

The optimum ratio is 80 parts antibody to 1 partantigen. There are specific terms for variations in thisratio.

Prozone - antibody excess:Antibodies saturating all

antigen sites; no antibodies forming cross-linkagesbetween 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 ofunagglutinated antigens


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False negative results DAT

All samples negative at the AHG phase should beincubated at room temperature for 5 minutes toachieve maximal sensitivity needed for complementdetection.

IAT Serum and/or rbcs lose reactivity if improperly stored.

Plasma used instead of serum can lead to failure todetect antibodies depending on presence of activecomplement (anti-Jka, -Jkb)

Temperature and incubation time affect attachmentof antibody or complement to cells.

An optimal proportion of serum to cells should beachieved: usually 2-3 drops serum to one drop of 5%cell suspension.


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False positive results: DAT and IAT ;

In specimens containing potent cold-reactiveantibodies agglutination may occur before adding theAHG reagent.

Dirty glassware may cause clumping of cells.

Over centrifugation

DAT

A positive DAT from a clotted sample should berepeated on an EDTA sample

Samples collected from infusion lines may havecomplement present on the cells.

IAT

Cells with a positive DAT will give a positive result in anyindirect antiglobulin procedure.


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COOMBS CELLS To show that test cells were properly washed and

that no neutralization or reagent deterioration hasoccurred, antibody-coated cells are used as apositive indicator.

In a negative antiglobulin test the anti-humanglobulin should remain active and this can be

demonstrated by the addition of IgG sensitized cells. Agglutination of the IgG sensitized cells after mixing

and centrifuging confirms that the anti-humanglobulin was added to the test, that the test cellswere properly washed and all free globulin molecules

were removed and that the anti-human globulin wasactive. Failure of the IgG sensitized cells to agglutinate

indicates that the original negative antiglobulin testresult is not valid and testing must be repeated.


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Lab 5 Coombs Tests