[LAB] AHG Testing

Question 1

One stage technique for detection of in vivo sensitization of RBC.

Answer 1

Direct Antiglobulin Test (DAT)

Question 2

Two-stage technique for detection of in vitro sensitization of RBC.

Answer 2

Indirect Antiglobulin Test (IAT)

Question 3

Identify the AHG reagent.

• polyvalent/broad spectrum
• has Ab to both human IgG and human C3d
• may bind kappa and lambda light chains

Answer 3

Polyspecific AHG

Question 4

Identify the AHG reagent.

• only 1Ab (either to human IgG or human C3b/C3d)
• specific for the Fc of the gamma heavy chain

Answer 4

Monospecific AHG

Question 5

How are polyclonal AHGs produced?

Answer 5

by immunizing 1 colony of rabbits with human IgG and another with human C3 Ag

Question 6

• Contains a mixture of Abs from different plasma cell clones
• Recognizes many epitopes

Answer 6

Polyclonal AHG

Question 7

How are monoclonal AHGs produced?

Answer 7

by immunizing laboratory mice with purified human IgG or by human C3 Ag

Question 8

• Contains Ab from 1 cell clone
• Recognizes a single epitope on an Ag

Answer 8

Monoclonal AHG

Question 9

What is the ratio of serum to cells?

Answer 9

40:1 (2 drops of serum to 1 drop of 5% RBC suspension)

Question 10

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

Answer 10

Albumin medium

Question 11

Identify the reaction medium.

▪ Enhances antibody uptake and allow incubation times to be decreased (10 – 15
minutes)
▪ Reduces the zeta potential surrounding the RBCs

Answer 11

Low ionic strength solution (LISS)

Question 12

Identify the reaction medium.

▪ Increased antibody uptake by removing water
molecules around the RBCs (the water of hydration theory), thereby increasing antibody concentration

Answer 12

Polyethylene glycol (PEG)

Question 13

What is the optimal temperature for incubation?

Answer 13

37℃

Question 14

How long is the incubation time when saline is used?

Answer 14

30 minutes to 2 hours

Question 15

How long is the incubation time when LISS or PEG is used?

Answer 15

10-15 minutes

Question 16

How many times must an RBC be saline-washed before adding AHG reagent?

Answer 16

3 times

Question 17

Ideally, the saline used for washing should be fresh and buffered to a pH of ________.

Answer 17

7.2 to 7.4

Question 18

Settings for centrifugation for reading.

Answer 18

1000 RCF for 20 seconds (500RCF for 15-20 seconds)

Question 19

What is the other name for the AHG test?

Answer 19

Coomb’s test

Question 20

What test is based on the principle that antihuman globulins obtained from immunized nonhuman species bind to human globulins such IgG or complement?

Answer 20

Antihuman globulin test

Question 21

What is the range of the normal saline solution used in direct AHG?

Answer 21

0.85-0.9% NaCl

Question 22

Enumerate the phases in the indirect AHG test.

Answer 22

1. Immediate spin phase
2. Incubation phase
3. AHG phase

Question 23

In a direct AHG test, what is the interpretation when red cells coated with IgG antibodies do not agglutinate directly when centrifuged?

Answer 23

Cells are sensitized with IgG or complement