[LAB] AHG Testing Flashcards
One stage technique for detection of in vivo sensitization of RBC.
Direct Antiglobulin Test (DAT)
Two-stage technique for detection of in vitro sensitization of RBC.
Indirect Antiglobulin Test (IAT)
Identify the AHG reagent.
- polyvalent/broad spectrum
- has Ab to both human IgG and human C3d
- may bind kappa and lambda light chains
Polyspecific AHG
Identify the AHG reagent.
- only 1Ab (either to human IgG or human C3b/C3d)
- specific for the Fc of the gamma heavy chain
Monospecific AHG
How are polyclonal AHGs produced?
by immunizing 1 colony of rabbits with human IgG and another with human C3 Ag
- Contains a mixture of Abs from different plasma cell clones
- Recognizes many epitopes
Polyclonal AHG
How are monoclonal AHGs produced?
by immunizing laboratory mice with purified human IgG or by human C3 Ag
- Contains Ab from 1 cell clone
- Recognizes a single epitope on an Ag
Monoclonal AHG
What is the ratio of serum to cells?
40:1 (2 drops of serum to 1 drop of 5% RBC suspension)
Identify the reaction medium.
▪ Allow antibody-coated cells to come into closer contact with each other so that agglutination occurs.
▪ Increases cell-to-cell contact
Albumin medium
Identify the reaction medium.
▪ Enhances antibody uptake and allow incubation times to be decreased (10 – 15
minutes)
▪ Reduces the zeta potential surrounding the RBCs
Low ionic strength solution (LISS)
Identify the reaction medium.
▪ Increased antibody uptake by removing water
molecules around the RBCs (the water of hydration theory), thereby increasing antibody concentration
Polyethylene glycol (PEG)
What is the optimal temperature for incubation?
37℃
How long is the incubation time when saline is used?
30 minutes to 2 hours
How long is the incubation time when LISS or PEG is used?
10-15 minutes
How many times must an RBC be saline-washed before adding AHG reagent?
3 times
Ideally, the saline used for washing should be fresh and buffered to a pH of ________.
7.2 to 7.4
Settings for centrifugation for reading.
1000 RCF for 20 seconds (500RCF for 15-20 seconds)
What is the other name for the AHG test?
Coomb’s test
What test is based on the principle that antihuman globulins obtained from immunized nonhuman species bind to human globulins such IgG or complement?
Antihuman globulin test
What is the range of the normal saline solution used in direct AHG?
0.85-0.9% NaCl
Enumerate the phases in the indirect AHG test.
- Immediate spin phase
- Incubation phase
- AHG phase
In a direct AHG test, what is the interpretation when red cells coated with IgG antibodies do not agglutinate directly when centrifuged?
Cells are sensitized with IgG or complement
