Antihuman Globulin Testing

Question 1

What is the purpose of antiglobulin testing?

Answer 1

Detect IgG or C3 b or d components on RBCs that do not directly agglutinate.

Question 2

One stage AHG procedure that detects in vivo sensitization

Answer 2

Direct Antiglobulin Test (DAT)

Question 3

Two stage AHG procedure that detects in vitro sensitization

Answer 3

Indirect Antiglobulin Test (IAT)

Question 4

Polyspecific reagents contain…

Answer 4

anti-IgG AND anti-complement

Question 5

Monospecific reagents contain

Answer 5

anti-IgG OR anti-C3b OR anti-C3d

Question 6

What antibodies are used to detect different epitopes on the same antigen?

Answer 6

Polyspecific antibodies

Question 7

What antibodies are used to detect a single epitope on an antigen?

Answer 7

Monospecific antibodies

Question 8

If a polyclonal DAT test is positive, what is the next step to take?

Answer 8

perform both anti-IgG and anti-complement monoclonal DATs

Question 9

IgG coated cells added to a negative AHG test are called what?

Answer 9

Check Cells

Question 10

How do Check Cells work?

Answer 10

Check cells are IgG coated cells that bind free AHG. They are added to a negative AHG test to validate the negative result making sure that the cells were washed properly and that the reagent was added and active.

Question 11

What type of blood cells are used to generate check cells and why?

Answer 11

Type O because they lack the A and B antigens. This makes sure that the patient’s anti-A and/or anti-B doesn’t interfere with the test.

Question 12

What is the test sample for a DAT?

Answer 12

Patient’s RBCs (washed)

Question 13

What is the test sample for an IAT?

Answer 13

Patient’s serum

Question 14

Which Ig is detected at the immediate spin?

Answer 14

IgM

Question 15

Which IgG is detected at the incubation stage?

Answer 15

IgG

Question 16

How does anti-human globulin (AHG) work?

Answer 16

bind to IgG bound to RBCs to bridge the zeta potential and making agglutination visible

Question 17

AHG reagents are derived from

Answer 17

rabbits and/or murine (mice)

Question 18

List sources of error that can result in a false positive AHG

Answer 18

polyagglutination
autoantibodies
antibodies to test components
contaminated sample
Sample storage
Contaminated glassware
faulty AHG
overcentrifugation
overreading
in vivo/vitro bacterial contamination

Question 19

List sources of error that can result in a false negative AHG

Answer 19

failure to add AHG 
improper washing
inactive AHG reagent
weak or heavy cell suspension
poor reading technique
Delay in testing
Altered incubation times
Amount of protein coating the RBCs

Question 20

What false-negative reactions can Check cells detect?

Answer 20

failure to add AHG reagent
improper cell washing
Inactive AHG reagent

Question 21

What are the two methods used to generate AHG reagents?

Answer 21

Conventional and Hybridoma

Question 22

Describe the Conventional method for obtaining a polyclonal AHG.

Answer 22

1. Rabbit is injected with pooled donor antigen
2. Rabbit forms antibody to that antigen
3. Antibodies are collected from multiple rabbits and purified

Final product: AHG Polyspecific polyclonal blend

Question 23

Describe the Hybridoma method for obtaining monoclonal antihuman globulin

Answer 23

1. Mouse is injected with human antigen (IgG in one mouse, complement in another)
2. Antibody-producing lymphocytes are collected from the spleen
3. spleen cells are fused with myeloma cells to make hybridoma cells
4. Clones are grown in tissue culture

Final product: Monoclonal antibodies (monoclonal IgG and monoclonal anti-complement can be pooled together to make an AHG polyspecific monoclonal blend)

Question 24

Why are spleen cells fused with myeloma cells in the hybridoma method for creating monoclonal AHG?

Answer 24

Myeloma cells can produce antibodies indefinitely