Antiglobulin test

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Antiglobulin test By John-Paul Adjah

Antiglobulin test By John-Paul Adjah

Antiglobulin test:

Antiglobulin test Also known as Anti Human Globulin test (AHG) or coombs test It is very important in detecting clinically significant unexpected antibodies either in-vivo or in-vitro

Principle of test:

Principle of test IgG coated red blood cells Complement coated red blood cells Red blood cells coated with incomplete IgG antibodies or complement do not readily agglutinate. These cells are said to be sensitized with IgG or complement

PowerPoint Presentation:

For agglutination to occur, an additional antibody that reacts with the FC portion of the IgG or the C3b or C3d must be added to system to bridge the antibodies or the C3bor C3d forming agglutination

Anti human globulin reagent production:

Anti human globulin reagent production It is made by injecting rabbits, sheep or goat with purified human IgG or C3. The animal is bled after a specified period and the reagent purified by absorbing unwanted antibodies The reagent may be polyspecific or monospecific Polyspecific Anti-human Globulin: contains a blend of Anti-IgG & Anti-C3b, -C3d and sometimes C4 Monospecific reagents: contains Anti-IgG alone or Anti-C3b,-C3d alone

Antiglobulin test and interpretation:

Antiglobulin test and interpretation Whether the cells have been coated, or sensitized, in vivo or in vitro the final interpretation is based on the following:

PowerPoint Presentation:

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

PowerPoint Presentation:

Antibodies are not attached to antigens during incubation 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 unagglutinated Agglutination following addition of CCC verifies negative result Negative antiglobulin test

Preparation of coombs control check cells (CCC):

Preparation of coombs control check cells (CCC) Positive Control : Sensitized 0 Rh (D) positive cells Negative Control : Sensitised 0 Rh (D) negative cells Unsensitised 0 Rh (D) positive cells

PowerPoint Presentation:

Take 0.5 ml of 5-6 times washed and packed 0 Rh (D) + ve red cells in a test tube. Add 2-3 drops of IgG anti-D (select a dilution ( titre 1:4) of anti-D which coats the red cells but does not agglutinate them at 37°C). Mix and incubate at 37°C for 30 minutes. If there is agglutination, repeat the procedure using more diluted anti-D. Wash 3-4 times and make 5% suspension in saline for use.

PowerPoint Presentation:

Perform a Direct antiglobulin test which should give a 2+ reaction. If no agglutination occurs, repeat the test by using less diluted anti-D serum. 0 Rh (D) negative sensitized red cells are also prepared by treating 0 Rh (D) negative cells in the same manner. The preparation should give a negative direct antiglobulin test (DAT)

False negative and positive results in antiglobulin test:

False negative and positive results in antiglobulin test False-negative reactions can occur when antigen-antibody reactions have occurred but WASHING IS INADEQUATE and free antibody remains when the anti-human globulin is added

PowerPoint Presentation:

False negatives results may be obtained when there is: 1. Inadequate cell washing 2. Delay in adding antiglobulin reagent after the washing step 3. Presence of small fibrin clots among the cells 4. Inactive, or forgotten antiglobulin reagent

Inadequate washing:

Inadequate washing Inadequate cell washing will lead to unbound antibody remaining in the red cell suspension that are available to neutralize the AHG (Coombs serum) so it will not react with red cells bound with antibody

Delay in adding AHG:

Delay in adding AHG Delay in adding Coombs serum after washing step will lead to antibody eluting off, detaching from the cell while cells are sitting in saline.  Now free antibody present in the saline neutralizes the AHG or Coombs reagent so it will not be able to react with the cells bound with antibody.

Presence of Small fibrin clot among cells:

Presence of Small fibrin clot among cells Small fibrin clot among the cells that were not washed away will have immunoglobulin and complement present.  The antibodies and complement in the fibrin clot neutralizes AHG leading to a negative test.

False positive results:

False positive results Using improper sample (clotted cells instead of EDTA for Direct Antiglobulin Test, DAT) Spontaneous agglutination (cells heavily coated with IgM) Non-specific agglutination ("sticky cells")

Types of Antiglobulin test:

Types of Antiglobulin test There are two types: a. Direct antiglobulin test (DAT) b. Indirect antiglobulin test (IAT)


DAT Direct antiglobulin test is used to detect in-vivo sensitization (coating) of red cells with immune antibody (IgG) or the complement component (C3b or C3d) in : 1. Diagnosis of haemolytic disease of the newborn (HDN) 2. Diagnosis of autoimmune haemolytic anemia (AIHA) 3. Investigation of haemolytic transfusion reaction (HTR) 4. Investigation of drug induced red cell sensitization eg . Penicillins, phenacetins, quinidine


Procedure ?


IAT The purpose of the indirect antiglobulin test is to detect In vitro sensitization of red cells.  This is done when sensitization does not lead to direct agglutination Uses Weak D's in donor bloods and pregnant females of individuals who type D (-) at room temperature 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.)



Factors affecting sensitivity of IAT:

Factors affecting sensitivity of IAT Temperature Optimal temperature : 37°C. Incubation at higher or lower temperature may give false results. Serum Cell ratio Increasing the ratio of serum to cells increases the antibody coating. Commonly used ratio in saline suspension is 2:1 but in LISS suspending cells, use equal volume of serum and 2% cell suspension. Incubation time Saline, Albumin or enzyme technique : 45-60 minutes LISS suspended cells - Routine 15 minutes Emergency : 5 minutes Suspension medium The sensitivity of IAT can be increased with addition of 22% bovine albumin, enzyme or by using LISS suspended cells.

Mechanism of enhancement:

Mechanism of enhancement LISS: reduces the ionic strength of the reaction medium thereby enhancing uptake of antibodies by the red cells Proteolytic enzymes: cleave sialoglycoprotein from red cells thereby reducing net charge between red cells Albumin: reduces the repulsion charges between red cells

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