Antihuman Globulin Testing Flashcards
What is the purpose of antiglobulin testing?
Detect IgG or C3 b or d components on RBCs that do not directly agglutinate.
One stage AHG procedure that detects in vivo sensitization
Direct Antiglobulin Test (DAT)
Two stage AHG procedure that detects in vitro sensitization
Indirect Antiglobulin Test (IAT)
Polyspecific reagents contain…
anti-IgG AND anti-complement
Monospecific reagents contain
anti-IgG OR anti-C3b OR anti-C3d
What antibodies are used to detect different epitopes on the same antigen?
Polyspecific antibodies
What antibodies are used to detect a single epitope on an antigen?
Monospecific antibodies
If a polyclonal DAT test is positive, what is the next step to take?
perform both anti-IgG and anti-complement monoclonal DATs
IgG coated cells added to a negative AHG test are called what?
Check Cells
How do Check Cells work?
Check cells are IgG coated cells that bind free AHG. They are added to a negative AHG test to validate the negative result making sure that the cells were washed properly and that the reagent was added and active.
What type of blood cells are used to generate check cells and why?
Type O because they lack the A and B antigens. This makes sure that the patient’s anti-A and/or anti-B doesn’t interfere with the test.
What is the test sample for a DAT?
Patient’s RBCs (washed)
What is the test sample for an IAT?
Patient’s serum
Which Ig is detected at the immediate spin?
IgM
Which IgG is detected at the incubation stage?
IgG
How does anti-human globulin (AHG) work?
bind to IgG bound to RBCs to bridge the zeta potential and making agglutination visible
AHG reagents are derived from
rabbits and/or murine (mice)
List sources of error that can result in a false positive AHG
polyagglutination autoantibodies antibodies to test components contaminated sample Sample storage Contaminated glassware faulty AHG overcentrifugation overreading in vivo/vitro bacterial contamination
List sources of error that can result in a false negative AHG
failure to add AHG improper washing inactive AHG reagent weak or heavy cell suspension poor reading technique Delay in testing Altered incubation times Amount of protein coating the RBCs
What false-negative reactions can Check cells detect?
failure to add AHG reagent
improper cell washing
Inactive AHG reagent
What are the two methods used to generate AHG reagents?
Conventional and Hybridoma
Describe the Conventional method for obtaining a polyclonal AHG.
- Rabbit is injected with pooled donor antigen
- Rabbit forms antibody to that antigen
- Antibodies are collected from multiple rabbits and purified
Final product: AHG Polyspecific polyclonal blend
Describe the Hybridoma method for obtaining monoclonal antihuman globulin
- Mouse is injected with human antigen (IgG in one mouse, complement in another)
- Antibody-producing lymphocytes are collected from the spleen
- spleen cells are fused with myeloma cells to make hybridoma cells
- Clones are grown in tissue culture
Final product: Monoclonal antibodies (monoclonal IgG and monoclonal anti-complement can be pooled together to make an AHG polyspecific monoclonal blend)
Why are spleen cells fused with myeloma cells in the hybridoma method for creating monoclonal AHG?
Myeloma cells can produce antibodies indefinitely
