ANTI HUMANGLOBULIN TEST

The test will detect IgG antibody that do not react in either saline or high protein media.
 DAT
 IAT

Principles of the Antiglobulin test

1. Antibody molecules and complement components are globulins.
2. Injecting an animal with human globulin stimulates the animal to produce antibody to the
foreign protein (i.e. AHG)
3. Human globulin will be agglutinated by AHG
4. Washed RBCs coated with human globulin are agglutinated by AHG
AHG reagents:
Polyspecific AHG
 Anti-IgG, Anti-C3b, Anti- C4b,C4d
Monospecific AHG
 Contains only one Antibody

Forms of Antihuman globulin

I. Direct Antiglobulin Test – used to demonstrate red blood cells that have been coated
(sensitized) with antibody in vivo.
Application:
1. Hemolytic disease of the newborn (HDN)
2. Hemolytic transfusion reaction
3. Autoimmune hemolytic anemia (AIHA) (auto-Abs coat own RBCs)
4. Diagnosis of red cell sensitization reaction caused by drugs.
Procedure of DAT:
1. Washed RBC + AHG reagent
2. Does not require incubation

Diseases that may cause a positive reaction to DAT

1. Viral pneumonia
2. Infectious Hepatitis
3. Infectious Mononucleosis
4. Megaloblastic anemia
5. Extreme Reticulocytosis
6. Acute Intermittent porphyria
7. Acute Hemolytic anemia
8. Lead poisoning

Drugs that may cause a positive DAT

1. Alpha-methyldopa
2. penicillin
3. cephalothin
4. phenlyhydrazine
5. quinine

II. Indirect Antiglobulin Test - Used to demonstrate the presence of free antibody in the patients serum.

Application:
1. Antibody detection
2. Antibody Identification
3. Red Cell phenotyping
4. Antibody titration

Procedure of IAT:
1. Incubate RBCs w/ anti sera
2. Wash 3x
3. Add AHG reagent
4. If negative, add check cells
 Result is valid if there is agglutination
 Result is invalid if there is no agglutination
Factors Affecting the Antiglobulin Test
 Cell: serum = 1:40 or 1gtt/2gtt
 Reaction medium
 Albumin
 LISS
 PEG
 Temperature (37C)
 Incubation time
 Washing
 Centrifugation
 1000RCF for 20 sec