ANTIHUMAN GLOBULIN TEST

2 MAJOR TYPES OF BLOOD GROUP ANTIBODIES

IgM

 Large pentamer structure

 Binds to corresponding antigen and directly agglutinate RBCs suspended in saline

IgG

 Monomer structure
 Some IgG antibodies are termed nonagglutinating or incomplete antibodies
 Too small to directly agglutinate sensitized RBCs

Adding AHG that contains anti-IgG to RBCs sensitized with IgG antibodies allows for
hemagglutination.

Also referred to as COOMB’S TEST

Can be used to detect RBCs sensitized with
 IgG alloantibodies
 IgG autoantibodies
 complement proteins

2 TYPES OF AHG TEST:

DIRECT ANTIGLOBULIN TEST (DAT)

 Detects in vivo sensitization

INDIRECT ANTIGLOBULIN TEST (IAT)

 Detects in vitro sensitization

AHG REAGENT

POLYSPECIFIC AHG

Contains anti-IgG and anti-C3b/C3d

MONOSPECIFIC AHG

Contains only one antibody specificity

 Anti-IgG
 Anti-C3b/C3d
PREPARATION OF AHG REAGENT

Involves injecting human serum or purified globulins into laboratory animals.

 Human globulin behaves as foreign antigen
 Laboratory animal will produce antibodies against the human globulins
Two types of antibody production
 Polyclonal AHG production
 Monoclonal AHG production

POLYCLONAL AHG PRODUCTION

Usually prepared using rabbits

Prepared by conventional technique
 Sheep or goats may be used when large volumes of antibody are required

Rabbits are injected with either complement or IgG

Antibody produced is collected and purified:
 Anti-IgG
 Anti-C3b/C3d

Monospecific antibodies are pooled together to form AHG Polyspecific reagents

MONOCLONAL AHG PRODUCTION

Usually prepared using mice

Employs the use of hybridoma technology
 Mice is injected with purified human globulin
 After a suitable immune response, spleen cells containing antibody-secreting
lymphocytes are harvested
 Spleen cells are combined with myeloma cells in the presence of PEG
 Spleen Cells + Myeloma Cells = HYBRIDOMA CELLS
 Clone cells that produce only one type of antibody

 Fused cells are placed in a HAT medium

 Hypoxanthine, Aminopterin, and Thymidine
 Only allows the growth of Hybridoma cells
DIRECT COOMB’S TEST (DAT)

Detects in vivo sensitization

Conditions that can result in in vivo sensitization of RBCS:
 Hemolytic Disease of the Newborn (HDN)
 Hemolytic Transfusion Reactions (HTR)
 Autoimmune Hemolytic Anemia (AIHA)
 Drug-induced Hemolytic Anemia

INDIRECT COOMB’S TEST (IAT)

Detects in vitro sensitization

Used in the following situations:
 Compatibility testing
 Antibody screening
 Determination of RBC phenotype
 Determination of Du (weak D)

CHECK CELLS

COOMB’S CELLS
 Group O RBCs sensitized with IgG
 Added to negative antiglobulin tests to validate negative AHG reactions
 After addition of O check cells, agglutination indicates:
 AHG reagent was added
 AHG reagent was not neutralized
 Lack of agglutination invalidates result

FACTORS AFFECTING AHG TESTING

Ratio of Serum to cells

 ↑ ratio = ↑ sensitivity of the test
 Minimum target ratio 40:1
 2 drops of serum
 1 drop of 5% RCS
REACTION MEDIUM
 Enhancement media must be used to enhance the agglutination reactions

1. ALBUMIN
 Makes use of 22% BSA (bovine serum albumin)
 Decreases incubation time from 60 minutes to 30 minutes.

2. LOW IONIC STRENGTH SOLUTIONS (LISS)

 Enhance antibody uptake by reducing the zeta potential between red cells
 Decreases incubation time from 30-60 mins to 10-15 mins
 3. POLYETHYLENE GLYCOL (PEG)
 Increases antibody uptake by removing water molecules surrounding the
RBCs
 Concentrating antibody

FACTORS AFFECTING AHG TESTING

Temperature

Rate of reaction of antibodies is optimal antibodies at 37°C

Incubation time

Saline = 30-120 mins

LISS/PEG = 10-15 mins at 37°C

Washing of RBCs

Cells must be washed at least 3 times before adding AHG reagent

Inadequate washing causes a false-negative reaction

Saline for washing

Buffered to a pH of 7.2-7.4

Addition of AHG

AHG should be added immediately after washing

Centrifugation for reading

Recommended method: 1,000 RCF for 20 seconds

↑ RCF = ↑ sensitivity
 Inadequate resuspension = false-positive
 Vigorous resuspension = false-negative