DAT & IAT/ABO Discrepancies Flashcards
Direct Antiglobulin Test
checking for sensitized RBC’s in vivo
Indirect Antiglobulin Test
Checking for sensitized RBC’s in vitro
When is the DAT ordered?
Hemolytic disease of the newborn investigation
Transfusion reaction investigation
Immune hemolytic anemia
Drug absorption
Weak d investigation
Antibody screen
Antibody identification panel
Crossmatch (if carried out through all phases)
Tests that commonly require an IAT
Polyspecific AHG
Contains both anti-IgG and anti-C3d.
Allows the reaction of agglutination to be seen for one or both of these antibodies
Monospecific AHG
Individual investigations can be performed to find whether an antibody on the RBC is causing the agglutination or the presence of complement is causing the agglutination
How can AHG be inactivated?
Free antibodies that are not attached to the RBC surface
What will happen if we have inactivated the AHG before it has an opportunity to interact with the antibodies on the surface of the RBCs?
False negative reaction and Coombs control (check cells) will be negative
What does a positive result indicate on the AHG control test (check cells)?
Free AHG was available to combine with the sensitized RBC’s (coombs control reagent) and cause agglutination
Do you perform a AHG control test if the tube has agglutination in the AHG phase of the test?
No, the AHG is reacting. We are checking for false negatives using the Coombs control cells
What does a negative result indicate on the AHG control test?
We don’t know where the AHG is, invalid test
An adult patient is found to have a positive direct antiglobulin test. How would you investigate?
1) check medications
2) elution, then antibody ID if caused by an antibody
3 causes of in vivo RBC sensitization
HDN, HTR, and AIHA
If a patient has autoimmune hemolytic anemia, what may be found on the patients RBC?
Autoantibody/complement
DAT positive, along with Rh control tube (possibly)
Common antibiotics that may absorb onto the RBC membrane that will give a positive DAT result
Penicillins
Cephalosporins
Erythromycin
Drugs that may cause an autoantibody mechanism that would give a positive DAT
Methyldopa Levodopa Mefinamic acid Procainamide Ibuprofen
Significance of a positive DAT result
Indicates that antibody has attached to the red cells in vivo
What phase does sensitization primarily occur in the IAT?
37 degrees C in vitro
Where does sensitization occur in the DAT?
In vivo
Zeta potential
Natural repulsion between RBCs. Caused by ionic charge on surface and surrounding RBCs
Where does AHG anti-IgG bind on the antibody molecule?
Fc portion
Where does AHG anti-complement bind on the complement molecules?
C3d
Primary usefulness of albumin
Allows Rh antibodies to appear at 37 phase - is not as broadly reactive as the others
Primary usefulness of LISS
Low ionic strength saline has broad use - but reaction’s primarily in AHG phase only of testing
Primary usefulness of PEG
Enhancement of reactions, some problems with interpretation, can’t read at 37 degree phase
Which potentiator increases the affinity of the antibody and antigen instead of lowering the zeta potential?
PEG
Which potentiator is used when the interpretation of the results is not done in the 37 degree phase of an IAT?
PEG
Order of complement
C1, C4, C2, C3, C5, C6, C7, C8, C9
Main purpose of AHG
Attaches to the surface of RBCs and spans the distance between RBCs to cause agglutination
Charge on the surface of a red blood cell
Net negative charge surrounding the RBC
What specifically is Coombs control (check cells)?
Sensitized RBC’s
What is the purpose of the AGH control (check cells) test?
Check to see if AHG has bridged between IgG antibodies or if the AHG is still free
Why would monospecific AHG (anti-IgG) most likely not react to an anti-M in the AHG phase?
Anti-M is an IgM, cold-reacting antibody
Why might polyspecific AHG react with an IgM antibody at the AHG phase and monospecific AHG will not?
Complement may have bound and left over compliment proteins are on the surface of the RBC
What would happen in a DAT test if compliment was bound to the RBC’s and polyspecific AHG was used?
False positive: compliment and anti-C3d
If a patient had an IgM antibody that was a broad thermal range and you were using monospecific AHG, where would the agglutination take place?
Positive IS and 37 phase, negative AHG phase
In an indirect or direct AHG test, what is reacting together to cause agglutination in the check cells step?
AHG and check cells
If a person had an autoimmune hemolytic anemia caused by an IgG, where would you expect to see a positive result - indirect AHG or direct AHG?
Direct
If a person had an IgG autoantibody, why wouldn’t you normally see a discrepancy in the reverse testing?
IgG is a warm reacting antibody - it wouldn’t bind to its antigen at room temp
How do you separate the antigen and antibody in a patient that has a positive DAT if you want to ID the antibody?
Elution
Possible outcomes of compliment binding
Hemolysis by lysis of RBC
Destruction of RBC by a macrophage recognizing compliment as an opsonin
What class of antibody is most likely identified in the AHG phase of testing?
IgG
Which compliment pathway is detected using polyspecific AHG?
Classical
