AHG Flashcards
ANTIHUMAN GLOBULIN TEST
Also known as the_____
Coombs’ test
AHG
Detects_____ antibodies and____ attached to red blood cells (RBCs).
IgG
complement proteins
REAGENTS
• These are derived from multiple clones of antibody-producing B cells, resulting in a mixture of antibodies that recognize different epitopes on the same antigen.
Polyclonal AHG Reagents:
REAGENTS
• These are secreted by a single clone of antibody-producing B cells, giving them a unique specificity for a particular epitope
Monoclonal AHG Reagents
• Produced by injecting an antigen into an animal (commonly a rabbit), which stimulates B cells to produce various antibodies against different parts of the antigen.
Polyclonal antigen
Production Process of Polyclonal Antibodies
1. Inject antigen into a rabbit – Stimulates the immune response.
2. Antigen activates B cells – B cells recognize the antigen and proliferate.
3. Plasma B cells produce different antibodies – These target multiple epitopes of the antigen.
4. Obtain antiserum from rabbit – The serum contains polyclonal antibodies that recognize various epitopes.
These antibodies are highly specific and produced using hybridoma technology, where a B cell is fused with a myeloma (cancer) cell to create an immortal antibody-producing cell line.
Monoclonal AHG
• Contains antibodies to human IgG and the C3d component of complement (some: anti-C3b, anti-C4b).
Polyspecific AHG
Contains only one antibody specificity, either anti-IgG or anti-C3d
Monospecific AHG
Components:
• Anti-IgG – Detects IgG-sensitized RBCs.
• Anti-C3d (and sometimes C3b, C4b) – Detects complement-coated RBCs.
Polyspecific AHG
• Used to determine whether RBC sensitization is due to IgG antibodies or complement.
Monospecific AHG
Types:
• Anti-IgG AHG – Binds only to IgG-coated RBCs.
• Anti-C3d AHG – Binds only to complement-coated RBCs.
Monospecific AHG
The AHG test is based on the fact that_____ are too small to directly cause RBC agglutination.
Instead, they coat the surface of RBCs in conditions like autoimmune hemolytic anemia (AIHA) or hemolytic transfusion reactions (HTR).
The AHG reagent contains anti-IgG antibodies that bind to these coated RBCs, causing visible agglutination.
IgG antibodies
There are two types of AHG tests
DAT
IAT
Detects in vivo sensitization of RBCs.
It identifies IgG antibodies, complement molecules, or both bound to a patient’s RBCs.
DIRECT AHG TEST (DAT)
A positive DAT suggests conditions like:
Hemolytic disease of the fetus and newborn (HDFN)
Hemolytic transfusion reactions (HTR)
Autoimmune hemolytic anemia (AIHA)
• In the_____ procedure, the patient’s RBCs are washed to remove unbound proteins, and AHG reagent is added.
• Agglutination indicates a positive result.
DAT
It detects in vitro sensitization of RBCs.
INDIRECT AHG TEST (IAT)
IAT
It is used in:
Pre-transfusion testing
Antibody screening
Antibody identification
Crossmatching
Antigen typing
In the_____, the serum is incubated with RBCs, washed, and AHG reagent is added.
lAT
• Agglutination indicates a specific reaction between an antibody in the serum and an antigen on the RBCs.
IAT
False negatives
Incomplete washing of RBCs
Insufficient AHG reagent added
Delayed result reading
Expired or improperly stored
AHG reagent
Low sensitivity of AHG reagent
Improper RBC suspension concentration
False positives
Overcentrifugation
Contaminated reagents or dirty glassware
Cold agglutinins or rouleaux formation
Improper use of controls
Store AHG at_____, use fresh reagents, perform quality control checks
2-8°C
Wash RBCs at least_____ with saline before adding AHG
3-4 times
Use____ suspension for optimal results
2-5% RBC
