Detection and
Identification
of Antibodies
Introduction
• The focus of antibody detection methods is on
“irregular” or “unexpected” antibodies.
• The unexpected antibodies of primary importance
are the immune alloantibodies.
• Naturally occurring antibodies may form as a result of
exposure to environmental sources.
• Passively acquired antibodies
• These antibodies are typically immunoglobulin G (IgG)
antibodies that react at 37°C or in the antihuman globulin
(AHG) phase of the indirect antiglobulin test.
• Autoantibodies, when present, may complicate the
detection of clinically significant antibodies.
Antibody Screen
• AABB Standards require the use of an antibody
screen to detect clinically significant antibodies in the
blood donor and the intended recipient.
• An antibody screen is included in standard prenatal
testing.
Antibody Screen: Tube Method
• The traditional method of detecting antibodies
• Serum or plasma mixed with RBCs having known
antigens
Antibody Screen: Tube Method
• 37°C incubation phase
• Enhancement media may be added to increase the degree
of sensitization
• Depending on the enhancement added, the tubes may be
centrifuged and observed for hemolysis and agglutination
following incubation
Antibody Screen: Tube Method
• Anti-human globulin reagent (AHG) phase
• Coombs’ Control Cells (also known as Check Cells)
Antibody Screen: Tube Method
RBC Reagents
• Each set of screen cells is accompanied by an antigen profile
sheet, detailing which antigens are present in each vial of cells.
• Antibodies that react more strongly with cells having
homozygous antigen expression are said to show dosage.
Antibody Screen: Tube Method
Enhancement Reagents (potentiators)
• May be added to the cell/serum mixture before
the 37°C incubation phase to increase the
sensitivity of the test system.
Antibody Screen: Tube Method
✓Antihuman Globulin (AHG) Reagents
➢Polyspecific AHG reagent
➢Monospecific AHG
Antibody Screen: Gel Method
• The screen cells used for this
technique meet the same
criteria as for the tube test but
are suspended in LISS to a
concentration of 0.8%.
Antibody Screen: Interpretation
• Agglutination or hemolysis at any stage of testing is a positive test result.
✓In what phase(s) did the reaction(s) occur?
✓Is the autologous control negative or positive?
✓Did more than one screen cell sample react? If so, did they react at the
same strength and phase?
✓Is hemolysis or mixed-field agglutination present?
✓True agglutination versus rouleaux?
Limitations to the Antibody Screen
• Antibody titer below the level of sensitivity
• Low-prevalence antigens and antibodies
• Lack of homozygous expression of the target antigen on
screening cells
Factors Influencing Sensitivity
Antibody Identification
• Testing against additional RBCs possessing
known antigens
• Reagents
• Antibody identification panel: a collection of 11 to
20 group O RBCs with various antigen expression
• Diverse pattern of antigen expression
• Should include cells with homozygous expression
ANTIBODY PANEL
Antibody ID Testing
• IS Phase
• 37°C Phase
IS
37
2+
0
0
0
0
0
2+
0
0
0
0
0
2+
0
0
0
2+
0
0
0
0
0
IAT Phase (or AHG)
IS
37
AHG
• Indirect Antiglobulin Test (IAT)
2+
0
0
0
0
0
0
0
0
2+
0
0
0
0
0
0
0
0
2+
0
0
0
0
0
2+
0
0
0
0
0
0
0
0
• To do this we use the Anti-Human Globulin
reagent (AHG)
• Wash cells 3 times with saline (manual or
automated)
• Add 2 drops of AHG and gently mix then spin
IS
LISS AHG
37°
2+
0
0
0
0
0
0
0
0
2+
0
0
2+
0
2+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
CC
You have agglutination…now what?
IS
LISS 37°
AHG
2+
0
0
0
0
0
0
0
0
2+
0
0
0
0
0
0
0
0
2+
0
0
0
0
0
2+
0
0
0
0
0
0
0
0
??
CC
1. Ruling Out
2+
0
0
2+
0
0
2+
0
2+
0
0
0
do NOT cross out heterozygous antigens that show dosage.
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2. Circle antigens not crossed out
2+
0
0
2+
0
0
2+
0
2+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3. Consider antibody’s usual reactivity
2+
0
0
2+
0
0
2+
0
2+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4. Look for a matching pattern
2+
0
0
2+
0
0
2+
0
2+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Rule of three
• The rule of three must be met to confirm the
presence of the antibody
• A p-value ≤ 0.05 must be observed
• This gives a 95% confidence interval
• How is it demonstrated?
3 Positive
cells
3 Negative
cells
2+
0
0
2+
0
0
2+
0
2+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Additional Techniques for Resolving
Antibody Identification
Selected Cell Panels
• The cells selected for testing should have minimal
overlap in the antigens they possess.
Additional Techniques for Resolving Antibody
Identification
Enzymes
• Ficin, papain, bromelin, or trypsin
• Modify the RBC surface by removing
sialic acid residues and by denaturing
or removing glycoproteins
Neutralization
• Substances used to
neutralize antibodies in
serum.
Adsorption
• Antigen/antibody complex composed of solid
precipitates that is removed by centrifugation
• Commercial reagents for adsorption
Autoadsorption
Homologous Adsorption
Differential Adsorption
Direct Antiglobulin Test and Elution
Techniques
• Detection of antibodies coating RBCs when investigating
suspected hemolytic transfusion reactions, HDFN, and
autoimmune and drug-induced hemolytic anemias
• Direct antiglobulin test (DAT)
• When IgG antibodies are detected, the next step is to dissociate
the antibodies from the RBC surface to allow identification.
• Elution techniques: total and partial
• Temperature-dependent methods
• pH adjustment
• Organic solvents
Antibody Titration
• Quantifies the amount of antibody present
✓Twofold serial dilutions of serum tested against a suspension of RBCs
possessing the target antigen
✓Clinical applications of antibody titration include:
Providing Compatible Blood Products
• Consideration of the frequency of the antigen in the population
and the clinical significance of the antibody
• When the patient sample contains clinically significant antibodies
or the patient has a past history of clinically significant antibodies,
units for transfusion must be antigen-negative.
• The crossmatch technique must demonstrate compatibility at
the AHG phase.
• Determination of the number of units that must be antigentyped to find a sufficient number to fill the crossmatch request.
• Proposal that certain populations, particularly sickle cell and
beta-thalassemia patients, receive units that are phenotypically
matched to reduce formation of alloantibodies.
THANK YOU!!! ☺