ANTIHUMAN GLOBULIN TEST Detects in vitro sensitization
ANTIHUMAN GLOBULIN TEST AHG REAGENT:
2 MAJOR TYPES OF BLOOD GROUP POLYSPECIFIC AHG
ANTIBODIES Contains anti-IgG and anti- IgM C3b/C3d
Large PENTAMER structure MONOSPECIFIC AHG
Binds to corresponding antigen and directly agglutinate RBCs Contains only one antibody suspended in saline specificity Anti-IgG IgG Anti-C3b/C3d MONOMER structure PREPARATION OF AHG REAGENT Some IgG antibodies are termed nonagglutinating or incomplete Involves injecting human serum or purified antibodies globulins into laboratory animals. Too small to directly agglutinate Human globulin behaves as foreign sensitized RBCs antigen NOTES: Laboratory animal will produce antibodies against the human Adding AHG that contains anti-IgG globulins to RBCs sensitized with IgG antibodies allows for TWO TYPES OF ANTIBODY hemagglutination. PRODUCTION
ANTIHUMAN GLOBULIN TEST Polyclonal AHG production
Monoclonal AHG production Also referred to as COOMB’S TEST Can be used to detect RBCs PREPARATION OF AHG REAGENT sensitized with 1. POLYCLONAL AHG PRODUCTION IgG alloantibodies IgG autoantibodies Usually prepared using RABBITS Complement proteins Prepared by conventional technique SHEEP OR GOATS may be 2 TYPES OF AHG TEST: used when large volumes of 1. DIRECT ANTIGLOBULIN TEST (DAT) antibody are required Rabbits are injected with either Detects in vivo sensitization complement or IgG Antibody produced is collected and purified: Anti-IgG Drug-induced Hemolytic Anti-C3b/C3d Anemia Monospecific antibodies are INDIRECT COOMB’S TEST (IAT) pooled together to form AHG Polyspecific reagents Detects in VITRO SENSITIZATION Used in the following situations: 2. MONOCLONAL AHG PRODUCTION Compatibility testing Usually prepared using MICE Antibody screening Employs the use of HYBRIDOMA Determination of RBC TECHNOLOGY phenotype Mice is injected with purified Determination of Du (weak human globulin D) After a suitable immune response, spleen cells CHECK CELLS containing antibody-secreting COOMB’S CELLS lymphocytes are harvested Spleen cells are combined with Group O RBCs sensitized with IgG myeloma cells in the presence of Added to Negative Antiglobulin PEG Tests to validate negative AHG Spleen Cells + Myeloma reactions Cells = HYBRIDOMA After addition of O check cells, CELLS agglutination indicates: Clone cells that produce only AHG reagent was added one type of antibody AHG reagent was not Fused cells are placed in a HAT neutralized medium Lack of agglutination invalidates Hypoxanthine, result Aminopterin, and Thymidine FACTORS AFFECTING AHG TESTING Only allows the growth of RATIO OF SERUM TO CELLS Hybridoma cells ↑ ratio = ↑ sensitivity of the test DIRECT COOMB’S TEST (DAT) Minimum target ratio 40:1 Detects in VIVO SENSITIZATION 2 drops of serum Conditions that can result in in vivo 1 drop of 5% RCS sensitization of RBCS: 1. ALBUMIN Hemolytic Disease of the Newborn (HDN) Makes use of 22% BSA (bovine Hemolytic Transfusion serum albumin) Reactions (HTR) Decreases incubation time from 60 Autoimmune Hemolytic minutes to 30 minutes. Anemia (AIHA) 2. LOW IONIC STRENGTH SOLUTIONS Vigorous resuspension = (LISS) false-negative
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
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