antiglobulin tes1

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AntiglobulintestBy John-Paul Adjah [email protected]



Antiglobulin test

• Also known as Anti Human Globulin test(AHG) or coombs test

•

It is very important in detectingclinically significant unexpectedantibodies either in-vivo or in-vitro



Principle of test

IgG coatedred bloodcells

Complementcoated red bloodcells

Red blood cellscoated withincomplete IgGantibodies orcomplement do

not readilyagglutinate.These cells aresaid to be

sensitized withIgG orcomplement



• For agglutination to occur, an additionalantibody that reacts with the FCportion of the IgG or the C3b or C3dmust be added to system to bridge theantibodies or the C3bor C3d forming

agglutination



Anti uman g o u in reagentproduction

• It is made by injecting rabbits, sheep or goatwith purified human IgG or C3.

• The animal is bled after a specified period and

the reagent purified by absorbing unwantedantibodies• The reagent may be polyspecific or

monospecific• Polyspecific Anti-human Globulin: contains a

blend of Anti-IgG & Anti-C3b, -C3d andsometimes C4

•

Monospecific reagents: contains Anti-IgG aloneor Anti-C3b,-C3d alone



Antiglobulin test andinterpretation

Whether the cells havebeen coated, or sensitized,in vivo or in vitro the final

interpretation is based onthe following:



Incomplete Antibodies

attach to the red cells

•Wash cells 3 to 4 times to remove any unattached antibodies

•Only antibodies attached with the cell remain

Add antihuman globulin

Visible agglutination in

test tube

Positive antiglobulin test



Antibodies arenot attached toantigens duringincubation

Wash cells three to four times to remove any unattached antibodies

Add anti human globulin

No visible agglutination

therefore a negative test

Add coombs control check cells (CCC)

Check cells

agglutinated whilst

original test cells

remained

unagglutinatedAgglutination following addition of CCC verifies negative result

Negative antiglobulin test



• Take 0.5 ml of 5-6 times washed and packed0 Rh (D) +ve red cells in a test tube.

• Add 2-3 drops of IgG anti-D (select adilution (titre 1:4) of anti-D which coats thered cells but does not agglutinate them at

37°C).

• Mix and incubate at 37°C for 30 minutes. Ifthere is agglutination, repeat the procedureusing more diluted anti-D.

• Wash 3-4 times and make 5% suspension in

saline for use.



• Perform a Direct antiglobulin test which

should give a 2+ reaction. If noagglutination occurs, repeat the test byusing less diluted anti-D serum.

• 0 Rh(D) negative sensitized red cellsare also prepared by treating 0 Rh(D)

negative cells in the same manner. Thepreparation should give a negativedirect antiglobulin test (DAT)



False negative and positiveresults in antiglobulin test

•

False-negative reactions can occur whenantigen-antibody reactions have occurredbut WASHING IS INADEQUATE and freeantibody remains when the anti-humanglobulin is added



• False negatives results may be obtainedwhen there is:

1. Inadequate cell washing

2. Delay in adding antiglobulinreagent after the washing step

3. Presence of small fibrin clots

among the cells4. Inactive, or forgotten

antiglobulin reagent



Inadequate washing

Inadequate cell washing will leadto unbound antibody remainingin the red cell suspension thatare available to neutralize the

AHG (Coombs serum) so it willnot react with red cells boundwith antibody



Delay in adding AHG

Delay in adding Coombs serum afterwashing step will lead to antibody

eluting off, detaching from the cellwhile cells are sitting in saline. Nowfree antibody present in the saline

neutralizes the AHG or Coombsreagent so it will not be able toreact with the cells bound with

antibody.



Presence of Small fibrin

clot among cells• Small fibrin clot among the cells

that were not washed away willhave immunoglobulin andcomplement present. The

antibodies and complement inthe fibrin clot neutralizes AHGleading to a negative test.



Types of Antiglobulin test

• There are two types:

a. Direct antiglobulin test (DAT)

b. Indirect antiglobulin test (IAT)

DAT



DAT• Direct antiglobulin test is used to detect in-

vivo sensitization (coating) of red cells withimmune antibody (IgG) or the complementcomponent (C3b or C3d) in :

1. Diagnosis of haemolytic disease of thenewborn (HDN)2. Diagnosis of autoimmune haemolytic anemia

(AIHA)

3. Investigation of haemolytic transfusionreaction (HTR)4. Investigation of drug induced red cell

sensitization

eg. Penicillins, phenacetins, quinidine



Procedure

IAT



IAT• The purpose of the indirect antiglobulin test is

to detect In vitro sensitization of red

cells. This is done when sensitization does notlead to direct agglutinationUses• Weak D's in donor bloods and pregnant

females of individuals who type D (-) at roomtemperature when doing ABO and Rh typing• Screening and identification of unexpected

(irregular) antibodies in serum.

• Compatibility testing• Detection of red cell antigens using specific

antibodies reacting only in antiglobulin test (K,Fya, Fyb, Jkb, etc.)



PROCEDURE



Factors affecting sensitivity ofIAT

TemperatureOptimal temperature : 37°C.Incubation at higher or lower temperature may give false results.

Serum Cell ratioIncreasing the ratio of serum to cells increases the antibody coating.

Commonly used ratio in saline suspension is 2:1 but in LISS suspendingcells, use equal volume of serum and 2% cell suspension.

Incubation timeSaline, Albumin or enzyme technique : 45-60 minutesLISS suspended cells - Routine 15 minutesEmergency : 5 minutes

Suspension mediumThe sensitivity of IAT can be increased with addition of 22% bovinealbumin, enzyme or by using LISS suspended cells.



Mechanism of enhancement

•

LISS: reduces the ionic strength of thereaction medium thereby enhancing uptakeof antibodies by the red cells

• Proteolytic enzymes: cleave sialoglycoproteinfrom red cells thereby reducing net chargebetween red cells

• Albumin: reduces the repulsion charges

between red cells

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