DAT Flashcards
Distinguish between IAT and DAT
IAT: in vitro, test what has attached to cell in test system
DAT: in vivo, test what has attached to the cell in the body
When is DAT ordered?
- Hemolysis present
- Positive auto-control
- Transfusion reaction workup
- Cordblood eval
DAT is predictive of ____ by immune process in 83% of the population exhibiting symptoms
Hemolytic anemia
Interpretations of positive auto-control
- DAT could be positive for IgG
- Transfused cells are in pt sample
- Autoimmune process
- Contamination of AHG procedure
- HDN/HTR
- Drug-induced Ab
Interpretation of negative auto-control
- DAT likely negative for IgG
- Ab not reactive with pt’s own cells
- Could be IgA or IgM or undetectable proteins
- Weak reaction, Ab dissociated from cell
- Complement could have destroyed cells before reaction
Principle of DAT
- Uses anti-human globulin (against Fc portion of antibody) to detect presence of Ab on the RBCs themselves
- No incubation step (no LISS inc)
Implications if check cells don’t work
Not enough washes
Inert control
Pt RBCs that you didn’t add AHG reagent to -> expect no agglutination bc cells shouldn’t be spontaneously clumping
AHG reagent
- polyspecific anti-human-globulin
- human/rabbit IgG + murine monoclonal anti-C3b, C3d (anti-complement)
Check cells in DAT
- Coomb’s control cells with anti-IgG
- Complement control with C3 attached
Why can failure to add AHG reagent quickly cause false negative?
Because complement/IgG can start to fall off the cell and dissolve in the plasma
In order to be a DAT, it must detect what?
Both complement and IgG on cell
Possible reasons for positive anti-C3b, C3d reaction
- HTR
- Drug-induced Ab
- Cold autoimmune anemia
- Complement activation due to bacterial infection or auto-Ab
Possible reasons for positive anti-IgG reaction
- HTR from allo-Ab
- HDN
- Drug-induced Ab
- Warm autoimmune anemia
PCH
Donath-Landsteiner IgG auto-Ab reacts with RBCs in the coldest parts of the body, which causes irreversible complement binding
Cold Agglutinin Syndrome (CAS)
Cold-reactive IgM auto-Ab binds complement as RBCs return to warmer part of the body, when IgM dissociates and leaves RBCs coated with complement
Warm Autoimmune Hemolytic Anemia (WAIH)
Auto-Ab reacts with patient’s RBCs in vivo, may or may not activate complement
Biggest reason to perform elution?
ONLY when positive DAT with anti-IgG!
Not positive DAT and anti-C3d! Need the anti-IgG!
Reasons to do elution
- Positive DAT and anti-IgG
- HTR workup
- HDN
- Confirm presence of specific Ag on RBCS, use in conj. with adsorption
- Confirm Ab specficity by showing it can be adsorbed onto RBC of only a particular blood group phenotype
Elution principle
Remove and ID Ab coating red cells
Elution steps
- Wash 20 drops of whole blood cells 4x
- Remove IgG from cell
- Make solution containing antibody testable for cells
- Perform Ab ID
In elution, what do you destroy and preserve?
Destroy cells, preserve Ab
Why do you save the supernatant from the last elution wash?
- To test it against screen or panel cells in the same method as eluate -> expect negative result
- Positive result = invalid
Interpretation of negative elution results
- Non-RBC Ab
- Low incidence IgG
- Weak IgG
Interpretation of “some positive” elution results
- Delayed HTR
- HDN
- New Ab formation
Interpretation of positive elution results
- Warm auto-Ab (most likely)
- HTR due to high incident Ab
- HDN due to high incident Ab
- Drug-induced Ab
Interpretation of mixed-field elution results
INVALID
You didn’t bring eluate to serological conditions or there’s interfering substances present
Interpretation of last wash positive in elution protocol
Inadequate washing, remake eluate
List elution methods
- Acidic
- Heat or freeze-thaw
- Organic solvents to reduce surface tension
T/F
Treatment of sensitized RBCS causes the dissociation of AB into the eluate
True
Which is done first?
elution or adsorption?
Adsorption
Who orders adsorption?
Blood banker only
Not clinician
Adsorption main purpose
Remove auto-Ab in order to ID underlying allo-Ab
Several purposes for adsorption
- Separate multiple Ab in plasma
- Confirm presence of specific Ag on RBCs (conj with elution)
- Confirm specificity of Ab by showing it can be adsorbed onto RBCs of only a particular blood group phenotype
Adsorption principle
Purposely coat red cells with Ab
Difference between elution and adsorption
Elution = take Ab off red cells
Adsorption = put Ab onto red cells
2 types of adsorption used to remove warm Ab and when you use them
- Autoadsorption: when patient has NOT been transfused
- Differential adsorption: when patient has been transfused
Differential adsorption
Adsorb patient plasma to 3 different known cell types
Adsorption controls
Test adsorbed plasma against patient cell (auto) or screen cells (differential) to make sure all Ab have been removed from plasma
T/F
Auto-control always indicates presence of auto-Ab
False
T/F
DAT may be positive with transfused cells only
True
Elution of transfused cells will indicate what?
Delayed HTR
