ANTIGLOBULINTEST o Contains only one antibody specificity, either anti-IgG or antibody to specific

complement components such as C3b or C3d

PRINCIPLE

Antihuman globulins (AHGs) obtained from immunized nonhuman species bind

to human globulins such as IgG or complement, either free in serum or attached
to antigens on red blood cells (RBCs).

HISTORY

1908 – Moreschi described the principle of the test. He used rabbit antigoat
serum to agglutinate rabbit RBCs that were sensitized with low nonagglutinating
doses of goat antirabbit RBC serum

1945 – Coombs and associates described the use of the antiglobulin test for the
detection of weak and nonagglutinating Rh antibodies in serum

1946 – Coombs and coworkers described the use of AHG to detect in vivo
sensitization of the RBCs of babies suffering from hemolytic disease of the
newborn

1947 – Coombs and Mourant demonstrated that the antibody activity that
detected Rh antibodies was associated with the anti-gamma globulin fraction in
the reagent

1951 – Dacie presented the first indication that there might be another antibody
activity present that influenced the final reaction.
PREPARATION OF AHG
1957 – Dacie and coworkers published data showing that the reactivity of AHG The classic method of AHG production involves injecting human serum or purified
to cells sensitized with warm antibodies resulted from anti-gamma globulin globulin into laboratory animals such as rabbits.
activity, whereas anti-nongamma globulin activity was responsible for the May be of polyclonal or monoclonal response
activity of cells sensitized by cold antibodies. o Polyclonal Antibodies
Mixture of antibodies from different plasma cell clones
o Monoclonal Antibodies
TYPES OF ANTIHUMAN GLOBULIN REAGENTS Derived from one clone of plasma cells
Polyclonal AHG Production
Polyspecific AHG o Usually prepared in rabbit. Sheep or goats for large volumes
o Different colonies are immunized with IgG and C3 antigens
o Contains antibody to human IgG and to the C3d component of human
complement. Other anticomplement antibodies, such as anti-C3b, anti-C4b, and o Uses serum from many donors
anti-C4d, may also be present o Both colonies are hyperimmunized
o The animals are bled if the antibody potency and specificity meets the predetermined
Monospecific AHG specifications
o Absorption with A1, B, and O cells
o Block titrations are performed
Monoclonal AHG Production
o Devised by Kohler and Milstein
o Immunization of mice with purified human globulin
o Mouse spleen cells are fused with myeloma cells
o Hybridomas are screened for antibodies with required specificity and affinity
o Propagated in tissue culture or by inoculation into mice

The reagent also contains buffers, stabilizers, and bacteriostatic agents and may be
dyed green for identification
 Reaction Medium
o Albumin
TWO (2) TYPES OF COOMBS TEST:
DIRECT ANTIGLOBULIN TEST o Low Ionic Strength Solution (LISS)
o A one-stage technique used to detect in vivo sensitization Enhances antibody uptake and allows incubation time to be decreased
Introduced by Low and Messeter
o Clinical applications:
o Polyethylene Glycol (PEG)
Hemolytic Disease of the Newborn
A water-soluble linear polymer
Hemolytic Transfusion Reaction
Effective on concentrating the antibody
Autoimmune and drug-induced hemolytic anemia
Anti-IgG is the reagent of choice
o Can detect 100 to 500 IgG molecules per RBC and 400 to 1,100 C3d molecules per
Temperature
RBC
o Optimal at 37 degrees Celsius
INDIRECT ANTIGLOBULIN TEST
o A two-stage technique used to determine in vitro sensitization of RBCs
Incubation Time
o Clinical Applications: o Saline – 30 to 120 minutes
Compatibility testing
o LISS or PEG – 10 to 15 minutes
Antibody Screen & Identification Washing of RBCs
RBC Phenotyping o Must be saline-washed for a minimum of three times before adding the AHG rgt
Saline for Washing
Titration of incomplete antibodies
o Should be fresh or buffered to a ph of 7.2-7.4
o A positive reaction is obtained when there are 100 to 200 IgG or C3 molecules on
Addition of AHG
the cell
o AHG should be added to the cells immediately after washing
o Tasks and its purposes: Centrifugation for Reading
Incubate RBCs with antisera o Centrifugation and resuspending the cells are crucial step in the technique
Allows time for antibody molecule attachment to RBC antigen
o CBER-recommended: 1000RCF for 20 seconds
Perform a minimum of three saline washes
Removes free globulin molecules o 500 RCF for 15 to 20 sec
Add antiglobulin reagent
Forms RBC agglutinates
Centrifuge
Accelerates agglutination by bringing cells closer together
Examine for agglutination
Interpret test as positive or negative
Grade agglutination reactions
Determines the strength of reaction
Add Check Cells
Check for neutralization of antisera by free globulin molecules

FACTORS AFFECTING THE ANTIGLOBULIN TEST

Ratio of serum to cells
o 40:1 (2 drops of serum and 1 drop of 5% RBC suspension)
 o Reverse ABO typing
Specific Gel
A specific reagent is incorporated into the gel
Antigen determination
Gel Low Ionic Antiglobulin Test (GLIAT)
AHG reagent is incorporated into the gel
Provides a safe, reliable, and easy-to-read AHG test
910 rpm for 10 minutes

MODIFIED AND AUTOMATED ANTIGLOBULIN TEST TECHNIQUES

Low Ionic Polybrene Technique
o Relies on low ionic conditions to rapidly sensitize cells with antibody
o Polybrene is a potent rouleaux-forming reagent
o Monospecific anti-IgG reagent must be used
Enzyme-Linked Antiglobulin Test
o The amount of color produced is measured spectrophotometrically and is
proportional to the amount of antibody present
o OD is measured at 405 nm
Solid Phase Technology

Gel Test
o Detects RBC antigen-antibody reactions by means of using a chamber filled with
polyacrylamide gel
o Three (3) types of gel tests:
Neutral Gel
Does not contain any specific reagent and acts only by its property of trapping
agglutinates
Applications:
o Antibody screening and ID with enzyme-treated or untreated RBCs