Ag-Ab Testing/AGT tests

Question 1

In terms of Ag-Ab testing in the lab, state 3 sources of antigens and 3 sources of antibodies, classifying each as a known or unknown source, as discussed in lecture.

Antigens:

Antibodies:

Answer 1

Antigens
Patient cells – unknown
Mfg. cells – known
Donor cells – unknown

Antibodies
Patient serum/plasma – unknown
Mfg. antisera – known
Donor serum/plasma – unknown

Question 2

Explain the difference between direct and indirect agglutination.

Answer 2

Direct agglutination

IgM is big enough to crosslink between two cells and cause direct agglutination
Considered a saline reaction – reaction is seen at IS

Indirect agglutination

IgG is not big enough to crosslink between two cells; therefore, it cannot cause direct agglutination – something must be added to aide in the agglutination process (Coombs reagent: anti-IgG that binds to IgG to facilitate agglutination of cells)

Question 3

Explain the principle of the antiglobulin test.

Answer 3

• Antibodies are globulins
• Anti-Abs bind to the Fc portion of the antibody and forms bridges between the antibody-coated RBCs resulting in agglutination

Question 4

Name three common blood groups that are capable of binding complement.

Answer 4

1. ABO
2. Lewis
3. Kidd

Question 5

Name four applications (causes of positive reactions) of the direct antiglobulin test.

Answer 5

1. Hemolytic Disease of the Fetus & Newborn (HDFN)
2. Transfusion Reactions (HTR)
3. Autoantibodies
4. Drugs

Question 6

Explain the reasons for each of the procedural steps in the IAT and DAT.

Answer 6

1. Incubate RBCs and serum (the only difference between the IAT and DAT – allows for cells to become sensitized)
2. Perform 3x washes (removes free globulin)
3. Add 2 drops of AHG (crosslinks Ag-Ab complexes)
4. Centrifuging (accelerates agglutination by bring cells close together)
5. Examining for agglutination (interprets test as a positive or negative)
6. Grade agglutination reaction (determines strength of reaction)
7. Add CCs to negative reactions (check for neutralization of AHG by free globulins)

Question 7

Name five applications of the indirect antiglobulin test.

Answer 7

1. Antibody Screen
2. Antibody ID
3. Compatibility Testing
4. Antibody Titration
5. Antigen Typing/Phenotyping

Question 8

Differentiate between an alloantibody and autoantibody in terms of:

1. Test that will indicate which category the antibody is in
2. Expected result seen for each

Answer 8

1. Test that will indicate which category the antibody is in
   Antibody identification (from the auto control)
2. Expected result seen for each
   Negative – indicating that the antibody is to a non-self antigen (alloantibody)
   Positive – indicating that the antibody is to a self-antigen (autoantibody)

Question 9

Compare and contrast the direct antiglobulin test and the indirect antiglobulin test with regard to:

1. purpose of the test
2. sample /specimen requirement

Answer 9

1. purpose of the test
   • DAT: detects in vitro sensitization
   • IAT: detects in vivo sensitization
2. sample /specimen requirement
   • DAT: patient cells or serum/plasma from EDTA or Red top tube
   • IAT: patient cells from an EDTA

Question 10

13. Classify each of the following situations as a source of false-positive and false-negative results in the antihuman globulin technique:
14. Too heavy or too weak of a cell suspension:
15. Incorrect incubation temperature:
16. Omission of patient serum, enhancement medium or AHG reagent:
17. Inadequate washing before the addition of AHG:
18. Neutralization of AHG reagent:
19. Interruptions or delays in the washing/ AHG steps:
20. Using cells with a positive DAT:
21. Over-reading of reactions:
22. Bacterial contamination of reagents:
23. Dirty glassware:

Answer 10

1. Too heavy or too weak of a cell suspension: false negative
2. Incorrect incubation temperature: false negative
3. Omission of patient serum, enhancement medium or AHG reagent: false negative
4. Inadequate washing before the addition of AHG: false negative
5. Neutralization of AHG reagent: false negative
6. Interruptions or delays in the washing/ AHG steps: false negative
7. Using cells with a positive DAT: false positive
8. Over-reading of reactions: false positive
9. Bacterial contamination of reagents: false positive
10. Dirty glassware: false positive